<table><caption>**Table S1: R-genes conservation in plants.** For each species investigated (lines), the number of annotated genes and super-R-genes as well as the number and percentage of orthologs (using as reference genomes rice for monocots and grape for eudicots) are indicated in columns. Fisher's Exact tests for the significant conservation of super-R-genes compared to the total number of annotated genes are provided in the last column (right).</caption><thead><tr><th rowspan="2">Species</th><th colspan="2">All genes</th><th colspan="2">Resistance genes</th><th rowspan="2">P-value</th></tr><tr><th>Genes #</th><th>Orth #</th><th>R-genes #</th><th>Orth #</th></tr></thead><tbody><tr><td>Sorghum</td><td>34008</td><td>6147</td><td>1717</td><td>413</td><td>2.52E-05</td></tr><tr><td>Maize</td><td>32540</td><td>4454</td><td>1867</td><td>319</td><td>5.23E-04</td></tr><tr><td>Brachypodium</td><td>25504</td><td>8533</td><td>1662</td><td>495</td><td>2.73E-02</td></tr><tr><td>Arabidopsis</td><td>33198</td><td>2389</td><td>1559</td><td>74</td><td>3.54E-04</td></tr><tr><td>Poplus</td><td>30260</td><td>4555</td><td>1297</td><td>122</td><td>2.68E-07</td></tr><tr><td>Papaya</td><td>19205</td><td>3199</td><td>703</td><td>101</td><td>1.81E-01</td></tr><tr><td>Soybean</td><td>46164</td><td>4013</td><td>3310</td><td>148</td><td>2.08E-17</td></tr><tr><td>Apple</td><td>58979</td><td>3498</td><td>4252</td><td>125</td><td>4.49E-17</td></tr><tr><td>Lotus</td><td>15470</td><td>1720</td><td>668</td><td>46</td><td>1.32E-03</td></tr><tr><td>Strawberry</td><td>34809</td><td>3289</td><td>1452</td><td>108</td><td>1.85E-02</td></tr><tr><td>Cacao</td><td>27814</td><td>4472</td><td>1439</td><td>149</td><td>1.47E-07</td></tr></tbody></table>
Paleo-evolutionary plasticity of plant disease
resistance genes

Rongzhi Zhang¹,², Florent Murat¹, Caroline Pont¹, Thierry Langin¹ and Jerome Salse¹*

**Abstract**

**Background:** The recent access to a large set of genome sequences, combined with a robust evolutionary scenario of modern monocot (i.e. grasses) and eudicot (i.e. rosids) species from their founder ancestors, offered the opportunity to gain insights into disease resistance genes (R-genes) evolutionary plasticity.

**Results:** We unravel in the current article (i) a R-genes repertoire consisting in 7883 for monocots and 15758 for eudicots, (ii) a contrasted R-genes conservation with 23.8% for monocots and 6.6% for dicots, (iii) a minimal ancestral founder pool of 384 R-genes for the monocots and 150 R-genes for the eudicots, (iv) a general pattern of organization in clusters accounting for more than 60% of mapped R-genes, (v) a biased deletion of ancestral duplicated R-genes between paralogous blocks possibly compensated by clusterization, (vi) a bias in R-genes clusterization where Leucine-Rich Repeats act as a 'glue' for domain association, (vii) a R-genes/miRNAs interome enriched toward duplicated R-genes.

**Conclusions:** Together, our data may suggest that R-genes family plasticity operated during plant evolution (i) at the structural level through massive duplicates loss counterbalanced by massive clusterization following polyploidization; as well as at (ii) the regulation level through microRNA/R-gene interactions acting as a possible source of functional diploidization of structurally retained R-genes duplicates. Such evolutionary shuffling events leaded to CNVs (i.e. Copy Number Variation) and PAVs (i.e. Presence Absence Variation) between related species operating in the decay of R-genes colinearity between plant species.

**Keywords:** R-genes, Duplication, Plasticity, Evolution

**Background**

Pathogen attacks from fungi [1], viruses [2], nematodes [3] or bacteria [4], compelled plants to prevent damages by engaging an “arms race” with these organisms. Therefore, plants have developed a battery of defense mechanisms involving (1) PTI (PAMP-Triggered Immunity) triggered by PAMP (Pathogen-Associated Molecular Patterns) [5-7] and (2) ETI (Effector-Triggered Immunity) triggered by effectors leading to hypersensitive response (referenced as HR [8]). Therefore, constant evolution leading to novel mechanisms is crucial for plant defense processes as well as adaptation to biotic stresses. The most studied disease resistance proteins encoding genes (hereafter R-genes) or genes involved in disease resistance pathways are Nucleotide-Binding-Sites (NBS) [9,10], Leucine-Rich

Repeats (LRR) [9,10], Toll-Interleukine1 Receptors (TIR), WRKY transcription factors [11,12], Lysine Motif (LysM) families [13,14], and Protein Kinase families (hereafter referenced as PKinase) [15,16]. R-genes can then be functionally classified into five distinct groups consisting in CNL (genes encoding proteins with coiled-coil, nucleotide binding site, leucine-rich repeat domains, i.e. CC-NBS-LRR), TNL (genes encoding proteins with Toll-interleukin receptor-like, nucleotide binding site, leucine-rich repeat domains, i.e. TIR-NBS-LRR), RLP (genes encoding proteins with receptor serine-threonine kinase like, extracellular leucine rich repeat domains, i.e. ser/thr-LRR), RLK (genes encoding proteins with kinase, extracellular leucine-rich repeat domains, i.e. Kin-LRR), and RGA (includes all other genes conferring resistance through different molecular mechanisms) classes [17]. LRR-RLK, LRR, LysM, LysM-kinase act as pattern-recognition receptors (PRR) involved in the PTI pathway, while NBS-LRR commonly responds in the frame of the

<footnote>* Correspondence: jsalse@clermont.inra.fr
¹INRA/UBP UMR 1095 GDEC 'Génétique, Diversité et Ecophysiologie des Céréales', 5 chemin de Beaulieu, 63100 Clermont-Ferrand, France
Full list of author information is available at the end of the article</footnote>
<table><caption>**Table S2: R-genes domains/family diversity in plants.** The numbers of different R-domain (lines) combinations are provided for each species (monocots and eudicots) investigated (columns) as well as for the R gene families structured into: ¹PRR/PTI (LRR-RLK, LRR, LysM, and LysM kinase, in blue), ²ETI (NBS-LRR, in red), ³other ‘R-combination’ (gene including NBS domain alone without LRR domains and RGA genes, in green), ⁴and ‘R-pathway’ (WRKYS, protein-kinases, in purple).</caption><thead><tr><th rowspan="2">DOMAINS</th><th colspan="4">Monocots</th><th colspan="6">Eudicots</th></tr><tr><th>Oryza sativa</th><th>Bra chyp odiu m dista chyo n</th><th>Sorgum hum bico lor</th><th>Zea may s</th><th>Vitis vinif era</th><th>Ara bido psis thali ana</th><th>Ca ric a pa pa ya</th><th>Frag aria vesc a</th><th>Glyc ine max</th><th>Lot us jap oni cus</th><th>Mal us x dom estic a</th><th>Pop ulus trich ocar pa</th><th>Theo bro ma caca o</th></tr></thead><tbody><tr><td>LRR¹</td><td>328</td><td>173</td><td>125</td><td>299</td><td>151</td><td>145</td><td>82</td><td>246</td><td>351</td><td>85</td><td>732</td><td>126</td><td>225</td></tr><tr><td>LRR-Pkinase¹</td><td>315</td><td>163</td><td>211</td><td>211</td><td>116</td><td>156</td><td>91</td><td>145</td><td>381</td><td>62</td><td>331</td><td>190</td><td>174</td></tr><tr><td>LRR-Pkinase-Ser/Thr Kinase¹</td><td>9</td><td>□</td><td>3</td><td>□</td><td>□</td><td>3</td><td>□</td><td>□</td><td>8</td><td>□</td><td>□</td><td>□</td><td>□</td></tr><tr><td>TIR-LRR-Pkinase¹</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td></tr><tr><td>TIR-LRR¹</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>□</td><td>24</td><td>□</td><td>1</td><td>4</td><td>□</td><td>□</td></tr><tr><td>TIR-LRR-WRKY¹</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>3</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td></tr><tr><td>LRR-WRKY¹</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>2</td></tr><tr><td>LysM¹</td><td>14</td><td>11</td><td>14</td><td>□</td><td>7</td><td>9</td><td>9</td><td>3</td><td>31</td><td>□</td><td>9</td><td>16</td><td>3</td></tr><tr><td>LysM-Pkinase¹</td><td>2</td><td>□</td><td>6</td><td>2</td><td>3</td><td>3</td><td>2</td><td>□</td><td>18</td><td>□</td><td>□</td><td>8</td><td>1</td></tr><tr><td>NBS-LRR²</td><td>235</td><td>98</td><td>176</td><td>45</td><td>121</td><td>53</td><td>19</td><td>52</td><td>215</td><td>9</td><td>420</td><td>76</td><td>86</td></tr><tr><td>NBS-LRR-Pkinase²</td><td>□</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>3</td><td>□</td><td>□</td></tr><tr><td>TIR-NBS-LRR-Pkinase²</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>2</td><td>□</td><td>□</td></tr><tr><td>NBS-LRR-Pkinase-WRKY²</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td></tr><tr><td>TIR-NBS-LRR²</td><td>2</td><td>□</td><td>□</td><td>2</td><td>32</td><td>78</td><td>8</td><td>11</td><td>142</td><td>8</td><td>145</td><td>21</td><td>5</td></tr><tr><td>NBS-LRR-WRKY²</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td></tr><tr><td>NBS³</td><td>298</td><td>100</td><td>105</td><td>91</td><td>65</td><td>19</td><td>23</td><td>75</td><td>50</td><td>43</td><td>224</td><td>23</td><td>122</td></tr><tr><td>NBS-WRKY³</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td></tr><tr><td>NBS-Pkinase³</td><td>1</td><td>□</td><td>1</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>2</td></tr><tr><td>TIR-NBS-Pkinase³</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td></tr><tr><td>TIR-NBS³</td><td>3</td><td>□</td><td>□</td><td>1</td><td>10</td><td>16</td><td>□</td><td>15</td><td>4</td><td>17</td><td>63</td><td>2</td><td>5</td></tr><tr><td>TIR-Pkinase³</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>3</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td></tr><tr><td>RG³</td><td>102</td><td>104</td><td>39</td><td>45</td><td>91</td><td>126</td><td>□</td><td>□</td><td>146</td><td>5</td><td>117</td><td>32</td><td>□</td></tr><tr><td>TIR³</td><td>5</td><td>2</td><td>2</td><td>9</td><td>7</td><td>31</td><td>5</td><td>91</td><td>20</td><td>23</td><td>124</td><td>8</td><td>4</td></tr><tr><td>Pkinase⁴</td><td>1018</td><td>930</td><td>872</td><td>1041</td><td>437</td><td>847</td><td>422</td><td>733</td><td>1603</td><td>381</td><td>1952</td><td>732</td><td>757</td></tr><tr><td>Pkinase-Ser/Thr Kinase⁴</td><td>205</td><td>□</td><td>65</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>162</td><td>□</td><td>□</td><td>□</td><td>□</td></tr><tr><td>Pkinase-WRKY⁴</td><td>1</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td><td>1</td><td>□</td><td>□</td></tr><tr><td>WRKY⁴</td><td>99</td><td>80</td><td>96</td><td>106</td><td>37</td><td>71</td><td>42</td><td>48</td><td>179</td><td>34</td><td>121</td><td>63</td><td>53</td></tr><tr><td>Total</td><td>2637</td><td>1662</td><td>1717</td><td>1854</td><td>1078</td><td>1559</td><td>703</td><td>1452</td><td>3310</td><td>668</td><td>4252</td><td>1297</td><td>1439</td></tr></tbody></table>
ETI pathway [18,19]. Finally, WRKY and protein-kinases, associated with protein domains encoded by R-genes (hereafter R-domains), can also be activated by PRRs in disease resistance pathways [18-20].

R-genes have been reported to be ancient and conserved
genes that have been detected in gymnosperms, plants
and animals to ensure immunity [21-23]. However,
comparative genomic analyses have shown that R-
genes are associated with a great structural diversity in
vertebrates and plants. For example, the presence of
TIR domains in conifers and mosses indicated that
TIR may represent an ancestral R-gene family with
shared functionality with their mammalian or insect
homologues regarding innate immunity [21,22,24-26].
TIR genes typically expanded in eudicot genomes,
while they have been reported to be absent (or at least
rare) in grass genomes [27-31]. Moreover, tandem and
segmental duplications have been reported as a source
of structural plasticity of NBS-LRR genes in plant
genomes [32]. Furthermore, PAV (Presence/Absence
Variation) polymorphisms often exist in a population
or between species [33-36]. Overall, small-scale stud-
ies (i.e. few R-genes families/domains and/or few plant
species investigated) have suggested R-genes as one of
the most plastic gene family in plants associated with
intense structural shuffling in the course of evolution
leading to synteny erosion or alternatively loss [37].
For example, evolutionary investigations of R-genes in
*Arabidopsis* and rice have been conducted suggesting
contrasted amplification of TNL and CNL families as
well as clusterization of NBS-LRRs *via* segmental and
tandem duplications or ectopic gene conversions [38].

Few studies have investigated the conservation of R-
genes across a large set of plant species and at the
whole-genome level. Genome sequences from flowering
plants that are derived from a common ancestor 135 to
250 million years ago (mya) are increasingly available
in the public domain for evolutionary studies. Recent
paleohistorical studies demonstrated that modern grass
genomes, including Panicoideae (sorghum [Sorghum
bicolor], [39] maize [Zea mays], [40]), Ehrhartoideae
(rice [Oryza sativa], [41]), and Pooideae (Brachypodium
distachyon; [42]), were shaped from n = 5 to 12 ancestral
grass karyotypes (AGKs) containing a minimal set of 6045
ordered protogenes with a minimum physical size of 33
Mb [43-45] through whole-genome duplication (WGD)
and ancestral chromosome fusion events. Likewise, the
recent comparison of numerous eudicot genomes (i.e.
mainly eurosids), including grape (Vitis vinifera; [46]),
poplar [47], *Arabidopsis thaliana* [48], soybean Glycine
max; [49], and cacao (Theobroma cacao; [50]), revealed
that modern eudicot genomes derived from an n = 7
ancestor that went through a paleohexaploidization event
to reach a n = 21 intermediate followed by numerous

lineage-specific WGDs and chromosome fusion events
[46,51]. During the last 135 to 250 million years of evo-
lution, the protein-coding gene families have been then
shaped by various gene duplication mechanisms, including
WGDs (or polyploidization), segmental duplications,
and tandem duplications. It is now well established that
all modern diploid plant species are highly shuffled
paleopolyploids [52-55].

Duplication (WGDs, segmental duplications and tandem duplications) were proposed as the major mechanisms driving R-genes family expansion or contraction from their traceable ancestral copies [56-58]. However, a systematic and detailed study of the paleohistorical evolution of R-genes across plant subfamilies including rosids species (Arabidopsis thaliana [48], Grape [59], Apple [60], Poplar [47], Soybean [49], Lotus [61], Strawberry [62], Cacao [50] and Papaya [63]), and grasses (Rice [41], Maize [40], Sorghum bicolor [39] and Brachypodium distachyon [42]) is still lacking. Particularly, how R-genes have behaved following polyploidization events is not well established. Such a precise investigation of the paleohistory of R-genes during the last 250 million years of evolution will unravel precise mechanisms that lead to the reduced conservation of R-genes observed between modern plant species.

Results

Disease resistance gene mapping, conservation and evolutionary patterns

To identify the largest set of plant R-genes, three comple-
mentary methods (see Methods section) were combined
(illustrated as Additional file 1: Figure S1) consisting in (1)
the detection of PFAM [64] domains, (2) the exploitation
of public genome annotations, and (3) the use of the Plant
Resistance Gene Database, PRGdb (http://prgdb.cbm.
fvg.it/index.php, [65]). The integration of the three pre-
vious approaches allowed to construct a non-redundant
set of putative R-genes in the plant species considered in
this study (Table 1, Additional file 1: Figure S2 and
Additional file 2: Dataset S1). Based on the genome anno-
tation approach, 2697 R-genes were identified in mono-
cots, and 3021 in eudicots, corresponding to a total set of
5718 plant R-genes (Table 1). Regarding the PFAM do-
main identification procedure, 8013 sequences in
monocots and 14996 in eudicot species were identified,
corresponding to 23009 putative R-genes sequences in
total (Table 1). Finally, the PRGdb aligned on the 13
genome sequences investigated unraveled 1874 sequences
in monocots and 2001 sequences in eudicot species.
These three complementary methods lead us to deliver
the most complete and non-redundant list in angio-
sperms consisting in 23641 R-genes sequences, 7883
for the monocots and 15758 for the eudicots (cf Table 1
and Additional file 2: Dataset S1). The identified R-genes
families deriving from the annotation, PFAM and PRGdb
<table><caption>**Table S3: Number of R-genes in the ancestral duplicated chromosomes in grasses.** The table provides the number of R genes and associated P-values (calculated by the permutation test method, see the material and methods) for the n=5/12 paleoancestor (AGK), rice (OS) Brachypodium (BD), sorghum (SB) and maize (ZM) genomes.</caption><thead><tr><th></th><th>N=5<br>AGK</th><th>N=12<br>AGK</th><th>P-value</th><th>OS</th><th>P-value</th><th>BD</th><th>P-value</th><th>SB</th><th>P-value</th><th>ZM</th><th>P-value</th></tr></thead><tbody><tr><td>A1<br>A5</td><td>108</td><td>72<br>40</td><td>8.09E-06</td><td>38<br>21</td><td>8.85E-03</td><td>32<br>14</td><td>3.54E-03</td><td>40<br>23</td><td>8.48E-03</td><td>49<br>25</td><td>6.85E-04</td></tr><tr><td>A8<br>A9</td><td>46</td><td>25<br>22</td><td>5.38E-01</td><td>16<br>12</td><td>3.81E-01</td><td>11<br>9</td><td>6.14E-01</td><td>17<br>8</td><td>3.70E-02</td><td>16<br>19</td><td>2.66E-01</td></tr><tr><td>A11<br>A12</td><td>70</td><td>39<br>43</td><td>4.85E-01</td><td>21<br>22</td><td>1.14E+00</td><td>12<br>5</td><td>7.55E-02</td><td>19<br>16</td><td>5.41E-01</td><td>18<br>23</td><td>1.78E-01</td></tr><tr><td>A2<br>A6</td><td>85</td><td>46<br>41</td><td>4.41E-01</td><td>22<br>24</td><td>1.31E+00</td><td>13<br>20</td><td>9.18E-02</td><td>29<br>22</td><td>2.33E-01</td><td>29<br>24</td><td>4.13E-01</td></tr><tr><td>A2<br>A4</td><td>47</td><td>13<br>36</td><td>4.00E-06</td><td>8<br>27</td><td>1.78E-05</td><td>8<br>15</td><td>4.57E-02</td><td>8<br>17</td><td>1.98E-02</td><td>9<br>21</td><td>4.06E-03</td></tr><tr><td>A3<br>A10</td><td>32</td><td>18<br>15</td><td>4.75E-01</td><td>17<br>8</td><td>5.31E-03</td><td>10<br>7</td><td>3.38E-01</td><td>10<br>7</td><td>3.85E-01</td><td>12<br>8</td><td>2.33E-01</td></tr><tr><td>A3<br>A7</td><td>77</td><td>44<br>36</td><td>2.02E-01</td><td>26<br>26</td><td>9.78E-01</td><td>21<br>24</td><td>3.04E-01</td><td>26<br>22</td><td>4.94E-01</td><td>25<br>26</td><td>4.25E-01</td></tr></tbody></table>
<table><caption>Table 1 R-genes catalog and conservation in plant genomes</caption><thead><tr><th rowspan="2">Species</th><th colspan="4">Genome data</th><th colspan="4">R-genes data set</th><th colspan="2">R-genes conservation</th></tr><tr><th>Nb chr.</th><th>size (Mbp)</th><th>Nb gene</th><th>Nb ortholog</th><th>Annot</th><th>Pfam</th><th>PRG db</th><th>Non-redundant R-genes*</th><th>OrthoSeq</th><th>%</th></tr></thead><tbody><tr><td><strong>Monocot</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Oryza sativa (rice)</td><td>12</td><td>372</td><td>41046</td><td>Ref</td><td>1180</td><td>2294</td><td>1316</td><td>2637(889;539;10;16;100;1544;214;102)</td><td>Ref</td><td>Ref</td></tr><tr><td>Sorghum bicolor</td><td>10</td><td>659</td><td>34008</td><td>6147(14,9%)</td><td>570</td><td>1616</td><td>159</td><td>1717(516;284;2;20;98;1158;68;39)</td><td>413(116;16;0;7;5;347;19;3)</td><td>24,1%</td></tr><tr><td>Zea mays (maize)</td><td>10</td><td>2365</td><td>32540</td><td>4454(10,85%)</td><td>480</td><td>2630</td><td>399</td><td>1867(558;140;13;15;106;1255;0;45)</td><td>319(104;6;2;3;3;261;0;6)</td><td>17,1%</td></tr><tr><td>Brachypodium distachyon</td><td>5</td><td>271</td><td>25504</td><td>8533(20,78%)</td><td>467</td><td>1473</td><td>nd</td><td>1662(435;199;2;11;80;1094;0;104)</td><td>495(137;19;0;4;7;417;0;1)</td><td>29,8%</td></tr><tr><td><strong>Monocot Total</strong></td><td>-</td><td>-</td><td><strong>133098</strong></td><td>-</td><td><strong>2697</strong></td><td><strong>8013</strong></td><td><strong>1874</strong></td><td>7883(2398;1162;27;62;384;5051;282;290)</td><td>1227(357;41;2;14;15;1025;19;10)</td><td>-</td></tr><tr><td><strong>Eudicot</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Vitis vinifera (grape)</td><td>19</td><td>302</td><td>21189</td><td>Ref</td><td>256</td><td>892</td><td>170</td><td>1078(421;229;50;10;37;557;0;91)</td><td>Ref</td><td>Ref</td></tr><tr><td>Arabidopsis thaliana</td><td>5</td><td>119</td><td>33198</td><td>2389(11,27%)</td><td>564</td><td>1407</td><td>1105</td><td>1559(436;168;125;12;73;1010;3;126)</td><td>74(27;0;0;1;0;58;0;2)</td><td>4,7%</td></tr><tr><td>Populus trichocarpa (poplar)</td><td>19</td><td>294</td><td>30260</td><td>4555(21,5%)</td><td>212</td><td>1229</td><td>134</td><td>1297(413;122;31;24;63;930;0;32)</td><td>122(53;4;1;4;6;89;0;2)</td><td>9,4%</td></tr><tr><td>Carica papaya</td><td>9</td><td>234</td><td>19205</td><td>3199(15,1%)</td><td>113</td><td>674</td><td>nd</td><td>703(200;50;13;11;42;515;0;0)</td><td>101(45;1;1;1;4;74;0;0)</td><td>14,4%</td></tr><tr><td>Glycine max (soybean)</td><td>20</td><td>949</td><td>46164</td><td>4013(18,94%)</td><td>1023</td><td>3104</td><td>318</td><td>3310(1097;411;166;49;179;2172;170;146)</td><td>148(51;0;1;3;8;114;6;3)</td><td>4,5%</td></tr><tr><td>Malus x domestica (apple)</td><td>17</td><td>742</td><td>58979</td><td>3498(16,51%)</td><td>853</td><td>4135</td><td>243</td><td>4252(1638;860;340;9;123;2292;0;117)</td><td>125(41;0;1;1;7;94;0;2)</td><td>2,9%</td></tr><tr><td>Lotus japonicus</td><td>6</td><td>500</td><td>15470</td><td>1720(8,12%)</td><td>nd</td><td>664</td><td>26</td><td>668(165;77;49;0;34;443;0;5)</td><td>46(18;1;0;0;2;36;0;1)</td><td>6,9%</td></tr><tr><td>Fragaria vesca (strawberry)</td><td>7</td><td>240</td><td>34809</td><td>3289(15,52%)</td><td>nd</td><td>1452</td><td>1</td><td>1452(483;154;148;3;52;884;0;0)</td><td>108(44;2;1;0;1;89;0;0)</td><td>7,4%</td></tr><tr><td>Theobroma cacao</td><td>10</td><td>430</td><td>27814</td><td>4472(20,1%)</td><td>nd</td><td>1439</td><td>4</td><td>1439(492;220;14;4;55;934;0;0)</td><td>149(55;3;0;1;10;113;0;0)</td><td>10,4%</td></tr><tr><td><strong>Eudicot Total</strong></td><td>-</td><td>-</td><td><strong>265899</strong></td><td>-</td><td><strong>3021</strong></td><td><strong>14996</strong></td><td><strong>2001</strong></td><td>15758(5345;2291;936;122;658;9737;173;517)</td><td>873(334;11;5;11;38;667;6;10)</td><td>-</td></tr></tbody></table>

*Number of non-redundant R-genes.

Nb, number.

Nd, not detected.

Numbers in bracket refers to LRR, NBS, TIR, LysM, WRKY, Pkinase, Ser/Thr and Disease resistance related genes respectively.

In bold, total non-redundant detected R-genes.

Ref, reference.
<table><caption>**Table S4: Number of R-genes in the recent duplicated maize chromosomes.** The table provides the number of R genes and associated *P*-values (calculated by the permutation test method, see the material and methods) for the maize duplicated regions deriving from the n=12 paleoancestor (AGK).</caption><thead><tr><th>AGK (n = 12) Chromosomes</th><th>Maize Chromosome</th><th>Maize R genes #</th><th>AGK (n = 12) R-genes #</th><th>Permutation P-value</th></tr></thead><tbody><tr><td rowspan="2">A1</td><td>m3</td><td>40</td><td rowspan="2">49</td><td rowspan="2">1.34E-05</td></tr><tr><td>m8</td><td>18</td></tr><tr><td rowspan="2">A5</td><td>m6</td><td>16</td><td rowspan="2">25</td><td rowspan="2">2.70E-01</td></tr><tr><td>m8</td><td>12</td></tr><tr><td rowspan="2">A8</td><td>m1</td><td>14</td><td rowspan="2">16</td><td rowspan="2">6.99E-03</td></tr><tr><td>m4</td><td>6</td></tr><tr><td rowspan="2">A9</td><td>m7</td><td>17</td><td rowspan="2">19</td><td rowspan="2">1.10E-02</td></tr><tr><td>m2</td><td>10</td></tr><tr><td rowspan="2">A11</td><td>m4</td><td>13</td><td rowspan="2">18</td><td rowspan="2">5.05E-02</td></tr><tr><td>m2</td><td>7</td></tr><tr><td rowspan="3">A12</td><td>m3</td><td>16</td><td rowspan="3">23</td><td rowspan="3">7.46E-02</td></tr><tr><td>m10</td><td>7</td></tr><tr><td>m1</td><td>3</td></tr><tr><td rowspan="2">A2</td><td>m5</td><td>22</td><td rowspan="2">29</td><td rowspan="2">2.55E-03</td></tr><tr><td>m4</td><td>10</td></tr><tr><td rowspan="3">A6</td><td>m9</td><td>16</td><td rowspan="3">24</td><td rowspan="3">1.61E-07</td></tr><tr><td>m5</td><td>6</td></tr><tr><td>m6</td><td>4</td></tr><tr><td rowspan="2">A2</td><td>m4</td><td>6</td><td rowspan="2">9</td><td rowspan="2">6.25E-01</td></tr><tr><td>m5</td><td>5</td></tr><tr><td rowspan="2">A4</td><td>m2</td><td>15</td><td rowspan="2">21</td><td rowspan="2">3.39E-01</td></tr><tr><td>m10</td><td>12</td></tr><tr><td rowspan="2">A3</td><td>m1</td><td>9</td><td rowspan="2">12</td><td rowspan="2">4.70E-02</td></tr><tr><td>m9</td><td>4</td></tr><tr><td rowspan="2">A10</td><td>m1</td><td>2</td><td rowspan="2">8</td><td rowspan="2">1.21E-02</td></tr><tr><td>m5</td><td>7</td></tr><tr><td rowspan="3">A3</td><td>m1</td><td>18</td><td rowspan="3">25</td><td rowspan="3">2.42E-02</td></tr><tr><td>m5</td><td>5</td></tr><tr><td>m9</td><td>5</td></tr><tr><td rowspan="2">A7</td><td>m2</td><td>8</td><td rowspan="2">26</td><td rowspan="2">1.02E-05</td></tr><tr><td>m7</td><td>24</td></tr></tbody></table>
approaches are detailed in Table 1 and Additional file 1:
Figure S2, showing that the total number of non-redundant
R-genes in plants is close to the dataset obtained with the
PFAM detection approach, suggesting that such method
is the most appropriate in delivering the largest and
most complete set of R-genes in any genomic sequence
of interest.

The conservation of R-genes between genomes was then
investigated using rice and grape as reference genomes
respectively for monocots and eudicots as they represent
the most closely related modern genome structures of the
reconstructed plant ancestral karyotypes [66]. The 2637
rice R-gene sequences were aligned against the three other
monocot species available, i.e. sorghum, *Brachypodium*
and maize. Similarly, the 1078 grape R-genes sequences
were aligned against the eight eudicot species investigated
(Table 1, 'Evolutionary Data' column). 413 orthologous
R-genes were identified in sorghum corresponding to
24.1% of the reference dataset (i.e. rice). Similarly, 319
orthologous R-genes were found in maize (17.1% of the
reference) and 495 in *Brachypodium* (29.8% of the ref-
erence). This result suggests that R-genes may appear
as more conserved than the total protein-coding genes,
with respectively 24.4%, 17.1%, and 29.8% of rice ortho-
logous R-genes characterized in sorghum, maize and
*Brachypodium* compared to 14.9%, 10.85%, and 20.78%
of overall annotated protein-coding genes conservation
observed for the same species (*P*-value < 5% Fisher
Exact Test, Additional file 1: Table S1). However, if the
protein kinase family is excluded, as it consists in the
largest (14788 kinases) one compared to the six others
(with an average of 1264 R-genes for these families),
the synteny conservation observed for R-genes is similar
to one observed for the total annotated protein-coding
genes. The R-genes synteny (excluding protein kinases)
at the chromosome level between grasses is illustrated
as Figure 1A. In contrast to grasses, in eudicot species
R-genes are significantly less conserved than the annotated
protein-coding genes (except for papaya) with 4.7%, 9.4%,
4.5%, 2.9%, 6.9%, 7.4%, and 10.4% orthologous R-genes
identified in *Arabidopsis*, poplar, soybean, apple, lotus,
strawberry, and cacao compared to 11.27%, 21.5%, 18.94%,
16.51%, 8.12%, 15.52%, and 20.10% for the conservation of
total protein-coding genes for the same species (*P*-value <
5% Fisher's Exact Test; Additional file 1: Table S1). This
result may suggest a reduced syntenic conservation of
R-genes in eudicots compared to monocots.

R-genes were classified into seven distinct groups,
according to their specific encoded protein domains
(R-domains). In monocots, we identified 2398 LRR, 1162
NBS, 27 TIR, 62 LysM, 384 WRKY, 5333 Protein-kinases,
and 290 RG. In eudicots, 5345 LRR, 2291 NBS, 936 TIR,
122 LysM, 658 WRKY, 9910 Protein-kinases, and 517
RG were characterized. The distribution of the R-domain

repertoire excluding Pkinases in the 13 plant species
investigated is illustrated as Figure 1B with a color code
that illuminates the six different R-domains (i.e. for a
total of 7743 LRR, 3453 NBS, 963 TIR, 171 LysM, 1042
WRKY and 807 RG). Regarding the six different R-domains
investigated, LRR and NBS are more abundant in the
investigated plant genomes (Figure 1B). LRR and NBS
consist in, on average, more than 50% and 20% of detected
R-genes in both eudicots and monocots respectively.
Few WRKY domains were detected in plants (~3.98% and
3.33% in monocot and eudicot species respectively).
However, the number of TIR domains appeared much
abundant in eudicots than the monocot species (Additional
file 1: Table S2) as previously reported [27-31], which may
indicated a specific amplification of such domain during
rosids paleohistory. The distribution of R-gene families
structured into PTI (consisting in LRR-RLK, LRR, LysM,
and LysM kinase), ETI (consisting in NBS-LRR), other Pat-
tern Recognition Receptors (PRRs) divided into R-domains
combinations (including NBS, TIR, RG, NBS-Pkinase,
NBS-WRKY, TIR-NBS, TIR-NBS-Pkinase, TIR-Pkinase,
hereafter 'R-combination') and genes involved in disease
resistance pathway (including WRKYS and protein-kinases,
hereafter 'R-pathway') is available as Additional file 1:
Figure S3. The observed distribution of R-gene families
in the investigated species from the more abundant
is R-pathway (13189) > PTI (5844) > R-combination
(2525) > ETI (2070).

In order to reconstruct the ancestral R-genes repertoire
in plants, we used the recently reconstructed ancestral
monocot (5 protochromosomes) and eudicot (7 proto-
chromosomes) karyotypes to investigate the R-genes
evolutionary dynamic. In Figure 1C, the evolutionary
scenario of the modern grass genomes deriving from a
n = 5 ancestor is illustrated [66]. Circular distributions
illuminated the conservation rate of R-genes families
excluding Pkinases (Figure 1C top) and their abundances
within the different species (Figure 1C bottom). In the
four grasses, the distribution of R-domains appeared very
similar, except for RG, more abundant in *Brachypodium*
compared to the other grasses investigated (Fisher
Exact Test *P*-value = 4.10E-08, 1.46E-09 and 7.38E-08
in comparison to rice, sorghum, and maize; illustrated
as red star in Figure 1C bottom). This phenomenon
can simply be explained by the differences in RGA
annotation and functional characterization efforts in
the different species investigated. Finally, WRKY appeared
less abundant in rice compared with the other grasses
(Fisher Exact Test *P*-value = 3.41E-04, 2.70E-05, 9.47E-07
in comparison to rice *Brachypodium*, sorghum, and maize;
illustrated as blue star in Figure 1C bottom). According to
the reconstructed five protochromosomes, dating back
to ~50-70 mya before the speciation of the four modern
species investigated (containing 1622, 732, 845, and 832
<table><caption>**Table S5: Number of R-genes clusters in ancient duplicated grass chromosomes.** The table provides for n=12 ancestral chromosomes (Chromosomes A1 to A12 in lines) the number of R genes (GN #, number of genes) and clusters (CLS #, number of clusters) observed in the paleoancestor (AGK), rice (OS) *Brachypodium* (BD), sorghum (SB) and maize (ZM) genomes.</caption><thead><tr><th>Chr</th><th>AGK</th><th>GN #</th><th>CLS #</th><th>OS</th><th>GN #</th><th>CLS #</th><th>BD</th><th>GN #</th><th>CLS #</th><th>SB</th><th>GN #</th><th>CLS #</th><th>ZM</th><th>GN #</th><th>CLS #</th></tr></thead><tbody><tr><td>A1</td><td>72</td><td>24</td><td>10</td><td>38</td><td>6</td><td>3</td><td>32</td><td>8</td><td>4</td><td>40</td><td>14</td><td>7</td><td>49</td><td>11</td><td>4</td></tr><tr><td>A5</td><td>40</td><td>16</td><td>5</td><td>21</td><td>4</td><td>2</td><td>14</td><td>6</td><td>3</td><td>23</td><td>11</td><td>3</td><td>25</td><td>8</td><td>3</td></tr><tr><td>A8</td><td>25</td><td>10</td><td>3</td><td>16</td><td>4</td><td>2</td><td>11</td><td>0</td><td>0</td><td>17</td><td>5</td><td>2</td><td>16</td><td>2</td><td>1</td></tr><tr><td>A9</td><td>22</td><td>13</td><td>5</td><td>12</td><td>4</td><td>2</td><td>9</td><td>6</td><td>3</td><td>8</td><td>4</td><td>2</td><td>19</td><td>6</td><td>3</td></tr><tr><td>A11</td><td>39</td><td>12</td><td>5</td><td>21</td><td>2</td><td>1</td><td>12</td><td>4</td><td>2</td><td>19</td><td>6</td><td>3</td><td>18</td><td>7</td><td>3</td></tr><tr><td>A12</td><td>43</td><td>13</td><td>6</td><td>22</td><td>4</td><td>2</td><td>5</td><td>2</td><td>1</td><td>16</td><td>6</td><td>3</td><td>23</td><td>6</td><td>3</td></tr><tr><td>A2</td><td>46</td><td>25</td><td>10</td><td>22</td><td>8</td><td>3</td><td>13</td><td>12</td><td>6</td><td>29</td><td>20</td><td>8</td><td>29</td><td>8</td><td>4</td></tr><tr><td>A6</td><td>41</td><td>12</td><td>4</td><td>24</td><td>4</td><td>2</td><td>20</td><td>5</td><td>2</td><td>22</td><td>9</td><td>4</td><td>24</td><td>8</td><td>4</td></tr><tr><td>A2</td><td>13</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>9</td><td>0</td><td>0</td></tr><tr><td>A4</td><td>36</td><td>7</td><td>3</td><td>27</td><td>2</td><td>1</td><td>15</td><td>3</td><td>1</td><td>17</td><td>0</td><td>0</td><td>21</td><td>4</td><td>2</td></tr><tr><td>A3</td><td>18</td><td>6</td><td>3</td><td>17</td><td>4</td><td>2</td><td>10</td><td>2</td><td>1</td><td>10</td><td>4</td><td>2</td><td>12</td><td>2</td><td>1</td></tr><tr><td>A10</td><td>15</td><td>8</td><td>4</td><td>8</td><td>2</td><td>1</td><td>7</td><td>2</td><td>1</td><td>7</td><td>2</td><td>1</td><td>8</td><td>6</td><td>3</td></tr><tr><td>A3</td><td>44</td><td>13</td><td>5</td><td>26</td><td>4</td><td>2</td><td>21</td><td>7</td><td>3</td><td>26</td><td>8</td><td>4</td><td>25</td><td>0</td><td>0</td></tr><tr><td>A7</td><td>36</td><td>14</td><td>6</td><td>26</td><td>2</td><td>1</td><td>24</td><td>10</td><td>5</td><td>22</td><td>6</td><td>3</td><td>26</td><td>8</td><td>4</td></tr></tbody></table>
A

<figure><img src="image_1.png" /></figure>

B

<figure><img src="image_2.png" /></figure>

C

<figure><img src="image_3.png" /></figure>

**Figure 1** R-genes conservation and evolution in plants. (A) Grass genome synteny is illustrated as concentric circles. The chromosomes are highlighted with a color code (right) that illuminates the n = 12 monocot ancestral genome structure (inner circle A1 to A12). Any radius of the circle shows orthologous chromosomes between *Brachypodium*, rice, sorghum, and maize genomes. Maize genome is depicted as a double circle originating from the maize-specific recent WGD. Colinear R-genes are linked with black lines between circles, and ancestral duplicated R-genes are linked with black lines at the center of the circle. (B) R-genes content from 13 plant genomes including monocots (rice, *Brachypodium*, sorghum, and maize) and eudicots (*Arabidopsis*, Grape, Cacao, Papaya, Strawberry, Poplar, Lotus, Apple, and Soybean). The color code (bottom) highlights the R-gene classes investigated (LRR, NBS, TIR, LysM, RG). (C) Evolutionary scenario of R-genes in monocots. The modern grass genome structures (bottom) are depicted with a five-color code that illuminates their relationship with the n = 5 (A5, A7, A11, A8, A4) and n = 12 (A1 to A12) ancestors (top), according to Murat et al. [67]. The characterized R-genes are illustrated as vertical bars on the chromosomes of modern and ancestral genomes. The percentages of R-gene classes (LRR, NBS, TIR, LysM, RG, highlighted with the color code legend at the bottom) are shown with circular distributions for the four monocot genomes (bottom), the rice/Brachypodium and sorghum/maize ancestral genome intermediates (center), as well as for the ancestral karyotype (top). Statistically enriched and impoverished R-gene families are illustrated respectively with red and blue dots on the circular distributions.
<table><caption>**Table S6: Number of R-genes clusters in recent duplicated maize chromosomes.** The table provides for n=12 ancestral chromosomes (Chromosomes A1 to A12 in lines) the number of R-genes (GN #, number of genes), R-genes in cluster (GNC #, number of genes in clusters) and clusters (CLS #, number of clusters) observed in maize (ZM) duplicated chromosomes genomes.</caption><thead><tr><th>AGK</th><th>chromosome</th><th>GN #</th><th>GNC #</th><th>CLS #</th></tr></thead><tbody><tr><td rowspan="2">A1</td><td>m3</td><td>40</td><td>14</td><td>7</td></tr><tr><td>m8</td><td>18</td><td>3</td><td>3</td></tr><tr><td rowspan="2">A5</td><td>m6</td><td>16</td><td>6</td><td>3</td></tr><tr><td>m8</td><td>12</td><td>5</td><td>3</td></tr><tr><td rowspan="2">A8</td><td>m1</td><td>14</td><td>3</td><td>2</td></tr><tr><td>m4</td><td>6</td><td>0</td><td>0</td></tr><tr><td rowspan="2">A9</td><td>m7</td><td>17</td><td>2</td><td>1</td></tr><tr><td>m2</td><td>10</td><td>2</td><td>1</td></tr><tr><td rowspan="2">A11</td><td>m4</td><td>13</td><td>10</td><td>5</td></tr><tr><td>m2</td><td>7</td><td>4</td><td>4</td></tr><tr><td rowspan="3">A12</td><td>m3</td><td>16</td><td>9</td><td>6</td></tr><tr><td>m10</td><td>7</td><td>7</td><td>6</td></tr><tr><td>m1</td><td>3</td><td></td><td></td></tr><tr><td rowspan="2">A2</td><td>m5</td><td>22</td><td>9</td><td>6</td></tr><tr><td>m4</td><td>10</td><td>3</td><td>2</td></tr><tr><td rowspan="3">A6</td><td>m9</td><td>16</td><td>15</td><td>10</td></tr><tr><td>m5</td><td>6</td><td></td><td></td></tr><tr><td>m6</td><td>4</td><td>0</td><td>0</td></tr><tr><td rowspan="2">A2</td><td>m4</td><td>6</td><td>1</td><td>1</td></tr><tr><td>m5</td><td>5</td><td>0</td><td>0</td></tr><tr><td rowspan="2">A4</td><td>m2</td><td>15</td><td>3</td><td>2</td></tr><tr><td>m10</td><td>12</td><td>4</td><td>3</td></tr><tr><td rowspan="2">A3</td><td>m1</td><td>9</td><td>2</td><td>1</td></tr><tr><td>m9</td><td>4</td><td>2</td><td>1</td></tr><tr><td rowspan="3">A10</td><td>m1</td><td>2</td><td>0</td><td>0</td></tr><tr><td>m5</td><td>7</td><td>6</td><td>3</td></tr><tr><td>m1</td><td>18</td><td>5</td><td>4</td></tr><tr><td rowspan="3">A3</td><td>m5</td><td>5</td><td>1</td><td>1</td></tr><tr><td>m9</td><td>5</td><td></td><td></td></tr><tr><td>m2</td><td>8</td><td>2</td><td>2</td></tr><tr><td>A7</td><td>m7</td><td>24</td><td>12</td><td>8</td></tr></tbody></table>
R-genes excluding Pkinase domains in rice, *Brachypodium*, sorghum, and maize respectively), we were able to re-construct a minimal founder (conserved) pool of 465 ancestral R-domains consisting in 361 LRR, 54 NBS, 6 TIR, 11 LySM, 42 WRKY, and 52 RG (Figure 1C top, Additional file 3: Dataset S2). Based on the same strategy, the evolutionary scenario of the eudicots has been used to unravel a minimal founder pool of 150 R-genes (Additional file 1: Figure S4). In order to understand in more details the evolution of the major PTI/ETI families, we have reconstructed their ancestral pools. The results suggests that PTI genes content in the ancestors are significantly higher than observed in each modern species (*P-value* = 6.33E-04, 3.98E-04, 3.37E-04 in grasses ancestor, rice-*Brachypodium* ancestor and sorghum-maize ancestor respectively, Additional file 1: Figure S5), while the ETI genes content is lower in ancestors compared to modern species (*P-value* = 1.12E-04, 5.03E-04, 3.97E-04 in grasses ancestor, rice-*Brachypodium* ancestor and sorghum-maize ancestor respectively). This result may suggest an opposite evolutionary trend between PTI and ETI families that are respectively lost and gained in the course of evolution.

R-genes plasticity in response to duplication events

We wanted to investigate the impact of polyploidy (or
whole genome duplication, hereafter WGD) in shaping
the modern R-genes repertoire deriving from a founder
pool of 465 (dating back from ~100 mya) and 150 (dating
back from ~250 mya) R-genes for respectively the grasses
and rosids. While massive duplicated gene deletion in the
course of evolution following WGD has been reported
in the literature [68,69], then leading to orthologous
dominant (i.e. retention of ancestral genes) and sensitive
(i.e. deletion of ancestral genes) blocks, the particular
evolutionary fate of R-genes in response of such diploi-
dization phenomenon is still not well established. To
understand the R-genes family plasticity in response to
WGDs, we used monocots as a model system to investigate
the retention of R-genes (excluding Pkinases) in duplicated
fragment pairs (Figure 2A). It has been shown that
protein-coding genes behave differently in response to
this diploidization process. Diploidization resistant genes
(i.e. gene functions retained as duplicates following WGDs)
are mainly transcription factors (TFs), transcription
regulators (TRs) as well as miRNAs to a less extent,
whereas the remaining gene families are considered as
diploidization sensitive in returning to a singleton status
after WGDs via selective gene deletion between dominant
and sensitive chromosomal blocks [70,71]. At the whole
genome level, we observed that only ~5% (25 out of 465)
ancestral R-genes mapped on the grass ancestor (n = 12)
were co-retained (i.e. paralogous genes observed in the
ancestral duplicated chromosome pairs), a much lower

rate than the one reported for transcription factors
(TF) as well as miRNA genes with up to 50% of observed
co-retention of ancestral duplicates [68,72]. Therefore,
the observed lower co-retention of ancestral paralogous
R-genes may suggest that R-genes act as diploidization
sensitive genes in returning to a singleton status after
WGD. However, for 71.43% (five out of seven) duplicated
chromosome pairs, the previous characterized deletion of
the diploidization sensitive R-genes does not follows the
subgenome dominance hypothesis (except for A1/A5 and
A2/A4; highlighted with red connecting lines in Figure 2A
bottom) in deriving dominant and sensitive blocks [68,73].
Instead, we observed that R-genes retention after WGD is
equally distributed between ancestral chromosome pairs
A8/9, A11/A12, A2/A6, A3/A7, and A3/A10 (permutation
test with *P-value* < 5%, Figure 2A and Additional file 1:
Table S3) in the four grass species. Intriguingly, after the
recent WGD in maize, for 64.29% (nine out of fourteen
paralogous pairs highlighted with red connecting lines
in Figure 2A bottom) of the duplicated chromosomes,
R-genes deletion was partitioned between the paralogous
pairs (permutation test with *P-value* < 5%, Additional file
1: Table S4 and Additional file 1: Figure S6).

Such observed absence in R-genes deletion partitioning
in grasses between ancestral duplicated chromosomes
may be due to R-gene clusters identified as more abun-
dant in ancestral sensitive chromosomes compared to
dominant chromosomal compartment (R² = 0.64 with
*P-value* = 4.73E-06 for sensitive chromosomes and
R² = 0.34 with *P-value* = 3.70E-03 for dominant chro-
mosomes; Figure 2B and Additional file 1: Table S5).
For example, between A2 (reported as dominant) and
A4 (reported as sensitive), there is no cluster located
in the A2 in contrast to seven genes in three clusters
on A4, while between A1 (reported as dominant) and
A5 (reported as sensitive) more clusters in the A5 (16
genes in five clusters) do not reverse or reduce the
reported dominance of A1 (24 genes in ten clusters).
In contrast, R-genes clusters in maize did not affected
the observed bias retention of duplicated R-genes between
paralogous fragments excluding for A5 (m6 vs m8), A12
(m3 vs m1/10), A2 (m4 vs m5) and A4 (m2 vs m10);
Figure 2A (bottom), Additional file 1: Table S6. This result
may indicate that the random deletion of R-genes after
WGD, not following the known subgenome dominance
rule for the ancestral tetraploidization, may be a conse-
quence of the high plasticity of such gene family evolving
particularly in local tandem duplications (also referenced
as clusterization in the next section) that may have
compensated the ancestral biased deletion of duplicates
in known sensitive subgenomes in the course of evolution.
However, for the recent WGD in maize dating back to
5 mya, tandem duplications or clusters can't offset the
dominance/sensitivity effect in such short period of
<table><caption>**Table S7: R-genes clusters distribution in plants.** OS, BD, SB, ZM, VV, TC, CP, FV, MD, PT, LJ, AT, and GM represent Rice, *Brachypodium*, Sorghum, Maize, Grape, Cacao, Papaya, Strawberry, Poplar, Lotus, *Arabidopsis*, and Soybean, respectively (columns). The number of clusters (Cluster #), the number of super-R-gene in clusters (super-R-gene clusters #), the total number of super-R-genes (Total super-R-genes #) and percentages are provided in lines. R-genes in cluster here also are divided into four categories including PTI, ETI, 'R-combination', and 'R-pathway'.</caption><thead><tr><th></th><th>OS</th><th>BD</th><th>SB</th><th>ZM</th><th>VV</th><th>AT</th><th>PT</th><th>CP</th><th>GM</th><th>MD</th><th>LJ</th><th>FV</th><th>TC</th></tr></thead><tbody><tr><td>Cluster #</td><td>486</td><td>366</td><td>374</td><td>405</td><td>179</td><td>328</td><td>172</td><td>142</td><td>665</td><td>857</td><td>137</td><td>292</td><td>255</td></tr><tr><td># R-gene in clusters #</td><td>1951</td><td>1214</td><td>1160</td><td>1139</td><td>776</td><td>1062</td><td>417</td><td>396</td><td>2397</td><td>3464</td><td>377</td><td>886</td><td>1011</td></tr><tr><td># PTI in clusters</td><td>486</td><td>231</td><td>240</td><td>301</td><td>208</td><td>196</td><td>105</td><td>98</td><td>543</td><td>890</td><td>88</td><td>272</td><td>299</td></tr><tr><td># ETI in clusters</td><td>205</td><td>77</td><td>144</td><td>36</td><td>146</td><td>117</td><td>42</td><td>24</td><td>317</td><td>500</td><td>12</td><td>38</td><td>77</td></tr><tr><td># R-combination in clusters</td><td>333</td><td>179</td><td>126</td><td>98</td><td>126</td><td>55</td><td>20</td><td>26</td><td>168</td><td>446</td><td>64</td><td>117</td><td>121</td></tr><tr><td># R-pathway in clusters</td><td>927</td><td>727</td><td>650</td><td>704</td><td>296</td><td>694</td><td>250</td><td>248</td><td>1369</td><td>1629</td><td>213</td><td>460</td><td>514</td></tr><tr><td>Total R-genes #</td><td>2637</td><td>1717</td><td>1867</td><td>1662</td><td>1078</td><td>1559</td><td>1297</td><td>703</td><td>3310</td><td>4252</td><td>668</td><td>1452</td><td>1439</td></tr><tr><td>Percentage (%)</td><td>73.99</td><td>70.70</td><td>62.13</td><td>68.53</td><td>71.99</td><td>68.12</td><td>32.15</td><td>56.33</td><td>72.42</td><td>81.47</td><td>56.44</td><td>61.02</td><td>70.26</td></tr></tbody></table>
<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

**Figure 2** R-genes conservation between duplicated blocks in grasses. (A) Illustration at the top of the grass ancestral genomes with n = 5 (A5, A7, A11, A8, A4) paleoduplicated into n = 12 (A1 to A12) defining 7 shared duplicated blocks (black arrows). The number of conserved R-genes (y-axis) between duplicated chromosomes (x-axis) in modern grasses is illustrated as box plots (ISTAT) illustrates non-significant differences; (P-value < 0.05, see Methods section). Distribution pattern of conserved R-genes in modern chromosome pairs is shown in rice, *Brachypodium*, sorghum and maize. (B) Illustration of the correlation between the number of observed R-genes in cluster (x-axis) and the total number of conserved R-genes (y-axis) characterized in the sensitive (red dot and curve) and dominant (black dot and curve) chromosomal blocks.
<table><caption>**Table S8: R-genes duplication frequency in maize.** A-The number of R-genes in singletons and clusters (columns) are indicated in lines for rice (OS), *Brachypodium* (BD), sorghum (SB), maize (ZM) characterized in R-loci, R-loci with Single R-gene duplication (corresponding to single-gene or small-scale duplication SSD), Single R-gene duplication Hotspots (where one R-loci region can be duplicated in other loci at least two times), % R-loci with single R-gene duplication (corresponding to the frequency of single R-gene duplication loci vs total R-loci), % Single R-gene duplication hotspots (corresponding to the frequency of hotspot loci vs total number single R-gene duplication loci) shown in lines. B-The relationship among the singleton R-locus, clustered R-locus, and hotspot R-locus was illustrated in the paleo-duplicated blocks mediated by whole genome duplication and transposed duplication events among the ancestral WGD chromosome pairs (R1 & R5) and non-paralogous chromosomes (R7). Blue, purple, and red rectangles represent genes, R-genes and duplicated genes respectively.</caption><thead><tr><th></th><th>OS (Singletons+Clusters)</th><th>BD (Singletons+Clusters)</th><th>SB (Singletons+Clusters)</th><th>ZM (Singletons+Clusters)</th></tr></thead><tbody><tr><td>R-loci</td><td>1173 (687+486)</td><td>815 (449+366)</td><td>931 (557+374)</td><td>1134 (729+405)</td></tr><tr><td>R-loci with Single R-gene duplication</td><td>153</td><td>74</td><td>115</td><td>404</td></tr><tr><td>Single R-gene duplication Hotspots</td><td>36</td><td>28</td><td>36</td><td>209</td></tr><tr><td>% R-loci with single R-gene duplication</td><td>13.04</td><td>9.08</td><td>12.35</td><td>35.63</td></tr><tr><td>% Single R-gene duplication hotspots</td><td>23.53</td><td>37.84</td><td>31.3</td><td>51.73</td></tr></tbody></table>

B

<figure><img src="image_1.png" /></figure>
time. Thus, our data led to the hypothesis that R-genes,
identified as diploidization sensitive genes, may have
followed the subgenome dominance hypothesis that was
compensate in the course of history by a reshuffling return
flow consisting in local tandem duplications, enriching
sensitive genomic compartments in R-genes content.

R-genes plasticity via clusterization and transposition
mechanisms

While R-genes have been reported to be clustered in grass chromosomes [34] based on few species or loci investigated, a large-scale investigation of this phenomenon is still lacking in monocots and dicots. The structural definition of a R-gene cluster was considered following a previous study where linked (i.e. clustered) R-genes were not interrupted by more than eight non-R-genes [74]. In Additional file 1: Table S7, we reported that there are about 69% and 63% R-genes on average organized in clusters in monocots and eudicots respectively, suggesting that R-genes families expanded by lineage-specific tandem duplications leading to duplicated gene copy variants associated with high sequence similarities. Surprisingly, in poplar for example, we detected only 32% of R-genes organized in clusters using the same strategy, then unraveling possible specific patterns of R-gene clusterization between species. The typology of the R-genes clusters differs between species then reinforcing the concept of a recent clusterization process with most (58% on average) of them consisting in clusters of two locally duplicated R-genes, especially in maize (72%), while the largest and rare cluster, made of six R-genes, was only observed in rice (Additional file 1: Figure S6).

The particular evolution of R-genes via clusterization was highly dynamic through lineage-specific rearrangements leading to the observed conservation/erosion of R-genes colinearity between grasses, referenced as Copy Number Variation (CNV) and Presence/Absence Variation (PAV). The Figure 3A illustrates an orthologous R-gene locus in grasses involving a 178 kb rice region on chromosome 1 (containing four R-genes in clusters), a 68 kb region on the *Brachypodium* chromosome 2 (a single R-gene), a 161 kb region of the sorghum chromosome 3 (a single R-gene), and the two orthologous regions in maize on chromosomes 3 (232 kb) and 8 (113 kb) both with no R-gene annotated. This example illustrates the extreme structural variation in R-gene content between orthologous regions. On the two maize paralogous regions no R-gene were identified, while transposable elements (TEs) are found with high concentration (green rectangles as shown in Figure 3A). Their presences suggest that TEs may be involved in the loss of R-genes through illegitimate recombination. The reconstruction of the evolutionary history of this locus illustrates the plasticity of the R-gene

family where a single ancestral R-gene is retained in
the modern *Brachypodium* and sorghum genomes but
evolved into CNV in rice (four copies) and PAV (no R-
gene) in maize.

The Figure 3B illustrates the retention of duplicated loci with 90% of sequence similarity in cacao, one locus with one R-gene (Tc06p002810 consisting TIR-NBS-LRR domains), and the duplicated locus harboring two R-genes (i.e. Tc03p00890 with a TIR domain and Tc03p00900 with NBS-LRR domains). We located precisely TSD (Target Site Duplication) motifs and long terminal reverse duplication, suggesting Tc06p002810 as the acceptor site (Figure 3B, Dotplot horizontal axis) and the duplicated region with two genes as the donor site (Figure 3B, Dotplot vertical axis). The acceptor site is characterized by a 7 kb fragmental deletion, which is located in the intergenic region of the donor site between Tc03p00890 and Tc03p00900 (with no transposable element or repeat detected in this particular fragment). The deletion of the intergenic fragment between the neighbor genes may have led to the read through of the ORFs leading the two neighbor genes fused into a single one (Tc06p002810) in the course of evolution. These paralogous regions from cacao may then suggest duplication as a major process resulting in domain shuffling (then reducing colinearity between species) between tandem duplicated R-genes.

Such R-gene structural plasticity may also be driven by
TEs as we illustrated in the Figure 3C with two tandem
duplicated R-genes with about 80% of sequence similarity
from the rice genome, i.e. Os06g16300 (LRR domain) and
Os06g16330 (LRR-Pkinase domains). Using LTR_Finder
[75], we identified a 240 bp ancient LTRs flanking the
Os06g16300 gene (dark blue arrow) as well as 5 bp TSD
motifs associated with 1.1 kb recent LTRs (light blue
arrow) flanking two transposase genes, i.e. Os06g16310
and Os06g16320. We then proposed an evolutionary
scenario for this locus, where Os06g16300 is the ancestral
gene (conserved with the modern *Brachypodium* gene,
Bradi1g43690) that have been partially (illustrated as red
exons) duplicated in tandem as Os06g16330 and finally
physically separated by the TE-based transposition of the
two transposases (Os06g16310 and Os06g16320). Overall,
this example of tandem duplication followed by TE-based
transposition events illustrates another source of R-gene
plasticity reported in the current analysis, leading to
R-gene synteny erosion between closely related species.

Taking into account the previous case examples ob-
tained from cacao and rice genomes and in order to in-
vestigate R-gene synteny erosion at the whole genome
level, we aligned the non-syntenic R-genes with the total
R-genes repertoire. Using the parameters CIP > = 70%
and CALP > = 70% [66] to identify the paired non-self
matches, and according to the similarity of flanked
protein-coding genes between the paired R-genes using
<table><caption>**Table S9: R-genes duplication frequency in maize compared to other grasses** The number of R-loci (singletons + clusters one cluster considered as one loci independently from the number of R-genes detected); Single R-gene duplication (number of non ortholog duplicated R-loci), % Single R-gene duplication % (Single R-gene duplication percentage) are provided in columns for rice (OS), *Brachypodium* (BD), sorghum (SB), maize (ZM). *P-values* (Fisher's Exact Test) are provided as last column for the comparison of ZM/OS, ZM/BD and ZM/SB.</caption><thead><tr><th>Species</th><th>R-loci</th><th>Single R-gene duplication</th><th>% Single R-gene duplication</th><th>Fisher's Exact Test</th></tr></thead><tbody><tr><td>ZM</td><td>1134</td><td>404</td><td>35.63</td><td rowspan="2">6.32E-24</td></tr><tr><td>OS</td><td>1173</td><td>153</td><td>13.04</td></tr><tr><td>ZM</td><td>1134</td><td>404</td><td>35.63</td><td rowspan="2">2.28E-29</td></tr><tr><td>BD</td><td>815</td><td>74</td><td>9.08</td></tr><tr><td>ZM</td><td>1134</td><td>404</td><td>35.63</td><td rowspan="2">1.71E-22</td></tr><tr><td>SB</td><td>931</td><td>115</td><td>12.35</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Figure 3** Different sources of R-genes plasticity in plants. (A) Evolutionary history of a locus located on the ancestral chromosome A5 showing R-gene conservation as well as CNV and PAV between rice, *Brachypodium*, sorghum and maize. Conserved genes, non-conserved genes, deleted genes and transposable elements are illustrated according to the legend at the bottom. Dotted black lines link orthologous genes between modern loci at the bottom. The ancestral gene content is illustrated at the top. (B) Illustration of an example of R-gene fusion in cacao after duplication. The dotplot illustrates the two copy-paste regions flanked by TSD (Target Site Duplication as black arrows) motifs and repeated sequence motifs (ctgaaaatg/attaaaatg). R-genes (TIR, NBS, LRR) classes are illustrated according to the color code at the bottom. (C) Illustration of a R-gene (Os06g16330) partially duplicated (Os06g16300) in rice separated by a transposition event (Os06g16310- Os06g16320). The reconstructed evolutionary scenario is illustrated based on a color code illuminating repeat (ancient, recent) and gene (duplicated, non-duplicated, transposase) content. (D) Duplicated R-gene cluster plasticity in maize. One central R-gene cluster (chromosome 1) consisting in R-genes (red) and non R-genes (grey) is duplicated on homeoelogous regions (chromosomes 1-5-7) with distinct R-gene contents.</figcaption></figure>
<table><caption>**Table S10: R-genes targeted by miRNAs in plants.** OS, BD, SB, ZM, VV, TC, CP, FV, MD, PT, LJ, AT, and GM represent Rice, *Brachypodium*, Sorghum, Maize, Grape, Cacao, Papaya, Strawberry, Poplar, Lotus, *Arabidopsis*, and Soybean, respectively; ¹Number of R-genes can be targeted by miRNAs; ²Percentage of resistant genes can be targeted by miRNAs; ³randomly selected 600 non-resistant genes ten times. ⁴Paired student t-test was performed on the percentage of genes targeted by miRNAs observed to the total R-gene vs non-R-genes.</caption><thead><tr><th rowspan="2">Species</th><th colspan="3">Total_R-genes</th><th colspan="3">NON_R-genes³</th></tr><tr><th>targets¹</th><th>R-genes</th><th>%²</th><th>targets¹</th><th>R-genes</th><th>%²</th></tr></thead><tbody><tr><td>OS</td><td>949</td><td>2637</td><td>35.99</td><td>676</td><td>6000</td><td>11.27</td></tr><tr><td>BD</td><td>476</td><td>1662</td><td>28.64</td><td>742</td><td>6000</td><td>12.37</td></tr><tr><td>SB</td><td>647</td><td>1717</td><td>37.68</td><td>662</td><td>6000</td><td>11.03</td></tr><tr><td>ZM</td><td>554</td><td>1867</td><td>29.67</td><td>676</td><td>6000</td><td>11.27</td></tr><tr><td>Average</td><td>656.50</td><td>1970.75</td><td>33.31</td><td>689</td><td>6000</td><td>11.48</td></tr><tr><td>VV</td><td>154</td><td>1078</td><td>14.29</td><td>892</td><td>6000</td><td>14.87</td></tr><tr><td>TC</td><td>631</td><td>1439</td><td>43.85</td><td>580</td><td>6000</td><td>9.67</td></tr><tr><td>CP</td><td>139</td><td>703</td><td>19.77</td><td>773</td><td>6000</td><td>12.88</td></tr><tr><td>FV</td><td>314</td><td>1452</td><td>21.63</td><td>1011</td><td>6000</td><td>16.85</td></tr><tr><td>MD</td><td>1565</td><td>4252</td><td>36.81</td><td>163</td><td>6000</td><td>2.72</td></tr><tr><td>PT</td><td>448</td><td>1297</td><td>34.54</td><td>420</td><td>6000</td><td>7</td></tr><tr><td>LJ</td><td>242</td><td>668</td><td>36.23</td><td>210</td><td>6000</td><td>3.5</td></tr><tr><td>AT</td><td>737</td><td>1559</td><td>47.27</td><td>799</td><td>6000</td><td>13.32</td></tr><tr><td>GM</td><td>1380</td><td>3310</td><td>41.69</td><td>2203</td><td>6000</td><td>36.72</td></tr><tr><td>Average</td><td>623.33</td><td>1750.89</td><td>35.60</td><td>783.44</td><td>6000</td><td>13.06</td></tr></tbody></table>

$$ \boxed{^4P = 7.34E-06} $$
E-value < e-10 as a blast threshold, we distinguished segmental from single-gene duplications (also referenced as Small-Scale Duplication *i.e.* SSD) from these gene pairs (detailed in Methods section). In rice, *Brachypodium*, sorghum, and maize, we found 13.04% (153 out of 1173), 9.08% (74 out of 815), 12.35% (115 out of 931), and 35.63% (404 out of 1134) R-genes loci (R-genes located in the same clusters were considered as a single locus) involved in single-gene duplications (Additional file 1: Table S8 and Additional file 4: Dataset S3), which is higher than the 5% to 7% of single-gene duplication frequency reported for the total annotated protein-coding genes in grasses [76]. Among grasses, the single-gene duplication frequency in maize is significantly higher than in rice, *Brachypodium*, and sorghum respectively (*P-value* = 6.32E-24, 2.28E-29, and 1.71E-22 in Fisher's Exact Test respectively, *cf* Additional file 1: Table S9). In addition, we observed hotspots of single-gene duplications where R-loci showing higher sequence similarity with at least two other non-related R-loci was considered as hotspot (Figure 3D). In maize, 51.73% (209 out of 404) of the single-gene duplications frequency was observed, a much higher rate compared to 23.53% (36 out of 153), 37.84% (28 out of 74), and 31.30% (36 out of 115) in rice, *Brachypodium*, and sorghum respectively (Additional file 1: Table S8). We can then speculate that the recent WGD in maize, dating back to 5 mya, may have promoted and accelerated R-gene singleton duplication frequency compared to the other grasses.

Homologous R-genes sequences within clusters gener-
ated by tandem duplications provided the structural
template to form novel R-gene informs though domain
recombinations. We characterized all the different R-
domains in modern clusters and observed a specific
domain affinity for clusterization (Additional file 1:
Figure S7A and Additional file 1: Table S2). NBS-LRR and
LRR-Pkinase combinations are observed as representing
the majority of domain combinations in clusters (on
average 31.78% and 42.63% out of the total R-domains in
clusters for NBS-LRR, 51.08% and 46.47% for LRR-Pkinase
domains, respectively in monocots and eudicots), compared
to rare observed combinations in clusters for LRR-NBS-
PKinase-WRKY or LRR-TIR-WRKY (Additional file 1:
Table S2). More interestingly, we observed a preference or
affinity in domain combinations where more than 90% of
them included LRR (Additional file 1: Figure S7B), with a
preferential observed R-domain association with Pkinase
(59% and 52% in monocots and eudicots respectively;
Additional file 1: Figure S7C) and NBS (41% and 46%
in monocots and eudicots respectively; Additional file 1:
Figure S7C) domains. Therefore, our data confirm and
largely refine previous conclusions suggesting LRR as a
'glue' for domain association leading to new combinations
of R-gene domains observed in modern species, one major

source of R-gene plasticity. This dynamic recombination
of R-domains within clusters, especially enriching NBS-
LRR associations, may promote the development a novel
source of disease resistance in the investigated species.

R-gene plasticity mediated by miRNA/R-gene interactome

MiRNAs, as a versatile class of post-transcriptional gene
regulator, are reported to be involved in a large variety
of cellular processes, including development and defense
responses in plants [77-79]. Small RNA cloning and
high-throughput sequencing from plants infected by
pathogens have shown that many microRNAs [80-82]
and siRNAs [83] may be involved in biotic defense
responses through up or down regulation of targeted
gene expression. We wanted then to investigate whether
the miRNA/R-gene interactome had an impact on the
R-genes evolutionary plasticity as the role of miRNAs
in plant immunity system has been largely reported in
the literature [80-82]. To unveil if the plant paleoevolution
has affected or even shaped the R-gene/miRNA interactome,
we investigated miRNAs potentially targeting R-genes in the
four monocots investigated (rice, *Brachypodium*, sorghum,
and maize) as well as in nine eudicots (grape, *Arabidopsis*,
strawberry, cacao, papaya, poplar, soybean, apple, and lotus).

We considered resistance genes as *in silico* targets of
miRNAs based on sequence mismatch scores using Tar-
getfinder algorithm [84] (detailed in Methods section).
On average, we characterized 33.31% and 35.60% R-genes
predicted as *in silico* targets of miRNA in monocots and
eudicots respectively, significantly higher than for non
R-genes with 11.48% and 13.06% respectively (*P-value* =
9.013e-05 in paired student t-test, Additional file 1:
Table S10). No highly significant differences where
observed between non-conserved and conserved R-genes
targeted *in silico* by miRNAs in monocot (Table 2). In
eudicots, these differences (*P-value* = 3.48E-02 between
conserved and non-conserved miRNA-targeted resistance
genes) are likely to be associated with the numerous
rounds of WGD. We observed a correlation (r = 0.7133
with *P-value* = 0.03) between the number of WGD rounds
and the number of *in silico* miRNA/R-gene interactions
that took place in the plant paleohistory (Figure 4A).
This observation may suggest that successive WGDs
may have increased or putatively shaped the R-gene/
miRNA *in silico* interactome. After recent WGDs, for
example, in soybean, ~50% of retained R-genes (Figure 4B)
are potential targeted by miRNAs with mismatch score
of <= 4. One explanation could be that additional species-
specific R-genes copies (deriving from lineage-specific
WGDs), then leading to R-gene functional redundancy, may
be repressed at the expressional level through miRNAs.
Such suggested impact of miRNA regulation on duplicated
R-genes expression may need to be biologically and
functionally validated.
<table><caption>**Table S11: R-domains targeted by miRNAs in eudicots.** Number and percentages of NBS, LRR, TIR, WRKY and Pkinase domains (columns) are provided for the nine eudicots species investigated. T-test (paired student t-test) was performed between the percentage of domain vs total R-gene regulated by miRNAs for NBS and LRR, TIR and LRR, LRR and WRKY, and LRR and Pkinase.</caption><thead><tr><th>Species</th><th>NBS</th><th>% NBS regulated by miRNA</th><th>TIR</th><th>% TIR regulated by miRNA</th><th>LRR</th><th>% LRR regulated by miRNA</th><th>WRKY</th><th>% WRKY regulated by miRNA</th><th>Pkinase</th><th>% Pkinase regulated by miRNA</th></tr></thead><tbody><tr><td>VV</td><td>229</td><td>25.76</td><td>50</td><td>24</td><td>421</td><td>19.24</td><td>37</td><td>5.41</td><td>557</td><td>12.03</td></tr><tr><td>AT</td><td>168</td><td>70.24</td><td>125</td><td>64</td><td>436</td><td>58.26</td><td>73</td><td>36.99</td><td>1010</td><td>46.93</td></tr><tr><td>PT</td><td>122</td><td>63.11</td><td>31</td><td>58.06</td><td>413</td><td>44.79</td><td>63</td><td>31.75</td><td>930</td><td>32.15</td></tr><tr><td>CP</td><td>50</td><td>32</td><td>13</td><td>23.08</td><td>200</td><td>19</td><td>42</td><td>14.29</td><td>515</td><td>18.64</td></tr><tr><td>GM</td><td>411</td><td>73.72</td><td>166</td><td>84.94</td><td>1097</td><td>54.79</td><td>179</td><td>34.64</td><td>2172</td><td>37.66</td></tr><tr><td>MD</td><td>860</td><td>70.58</td><td>340</td><td>71.76</td><td>1638</td><td>47.01</td><td>123</td><td>19.51</td><td>2292</td><td>28.1</td></tr><tr><td>LJ</td><td>77</td><td>74.03</td><td>49</td><td>71.43</td><td>165</td><td>40.61</td><td>34</td><td>23.53</td><td>443</td><td>31.38</td></tr><tr><td>FV</td><td>154</td><td>17.53</td><td>148</td><td>16.89</td><td>483</td><td>20.5</td><td>52</td><td>23.08</td><td>884</td><td>23.53</td></tr><tr><td>TC</td><td>220</td><td>81.36</td><td>14</td><td>71.43</td><td>492</td><td>50.2</td><td>55</td><td>32.73</td><td>934</td><td>37.58</td></tr><tr><td>Average</td><td>254.56</td><td>56.48</td><td>104.00</td><td>53.95</td><td>593.89</td><td>39.38</td><td>73.11</td><td>24.66</td><td>1081.89</td><td>29.78</td></tr></tbody></table>

$P = 2.17E-03$ $P = 8.04E-03$

$P = 1.21E-03$ $P = 3.97E-03$
<table><caption>Table 2 miRNA repertoire targeting conserved and lineage-specific R-genes in plants</caption><thead><tr><th rowspan="2">Lineage-specific WGD rounds</th><th colspan="4">Conserved<sup>1</sup></th><th colspan="3">Non-conserved<sup>2</sup></th></tr><tr><th>Species</th><th>Targets<sup>3</sup></th><th>Total orth<sup>4</sup></th><th>(%)<sup>5</sup></th><th>Targets<sup>3</sup></th><th>Total non-orth<sup>4</sup></th><th>(%)<sup>5</sup></th></tr></thead><tbody><tr><td><strong>Monocot</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0 WGD</td><td>OS</td><td>178</td><td>521</td><td>34.17</td><td>771</td><td>2116</td><td>36.44</td></tr><tr><td>0 WGD</td><td>BD</td><td>156</td><td>495</td><td>31.52</td><td>319</td><td>1167</td><td>27.34</td></tr><tr><td>0 WGD</td><td>SB</td><td>158</td><td>413</td><td>38.26</td><td>489</td><td>1304</td><td>37.50</td></tr><tr><td>1 WGD</td><td>ZM</td><td>105</td><td>319</td><td>32.92</td><td>448</td><td>1548</td><td>28.94</td></tr><tr><td><strong>Eudicot</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0 WGD</td><td>VV</td><td>29</td><td>282</td><td>10.28</td><td>124</td><td>796</td><td>15.58</td></tr><tr><td>0 WGD</td><td>TC</td><td>77</td><td>149</td><td>51.68</td><td>554</td><td>1290</td><td>42.95</td></tr><tr><td>0 WGD</td><td>CP</td><td>22</td><td>101</td><td>21.78</td><td>116</td><td>602</td><td>19.27</td></tr><tr><td>0 WGD</td><td>FV</td><td>39</td><td>108</td><td>36.11</td><td>275</td><td>1344</td><td>20.46</td></tr><tr><td>1 WGD</td><td>MD</td><td>49</td><td>125</td><td>39.20</td><td>1516</td><td>4127</td><td>36.73</td></tr><tr><td>1 WGD</td><td>PT</td><td>59</td><td>122</td><td>48.36</td><td>389</td><td>1175</td><td>33.11</td></tr><tr><td>1 WGD</td><td>LJ</td><td>23</td><td>46</td><td>50.00</td><td>219</td><td>622</td><td>35.21</td></tr><tr><td>2 WGD</td><td>AT</td><td>51</td><td>74</td><td>68.92</td><td>685</td><td>1485</td><td>46.13</td></tr><tr><td>2 WGD</td><td>GM</td><td>72</td><td>148</td><td>48.65</td><td>1307</td><td>3162</td><td>41.33</td></tr></tbody></table>

Note: OS, BD, SB, ZM, VV, TC, CP, FV, MD, PT, LJ, AT, and GM represent Rice, *Brachypodium*, Sorghum, Maize, Grape, Cacao, Papaya, Strawberry, Poplar, Lotus, *Arabidopsis*, and Soybean, respectively; ¹Conserved R-genes are associated with orthologous genes with other monocots and eudicots species; ²Non-conserved R-genes do not have orthologous genes with other species either monocots and eudicots; ³Number of R-genes targeted by miRNAs; ⁴Number of total orthologous genes; ⁵Percentage of R-genes targeted by miRNAs.

Finally, we investigated the R-domains/miRNA affinity and observed that NBS/TIR > LRR > WRKY/PKinase domains are preferentially targeted by miRNAs (P-value = 2.17E-03 and 8.45E-03, 1.21E-03, and 3.97E-03 with paired student t-test for NBS vs LRR, TIR vs LRR, LRR vs WRKY, LRR vs Pkinase, respectively; Figure 4C & Additional file 1: Table S11). We also observed that 67% and 63% of R-genes clusters are targeted by miRNA in contrast to 33% and 37% of singleton R-genes, respectively in eudicots and monocots (Additional file 1: Figure S8 and Additional file 1: Table S12). Overall, the interaction affinity between species-specific R-genes (in clusters and in majority involving NBS/TIR domains) and miRNAs after WGD can be considered as a major source of R-gene family plasticity in plants, as part of a possible functional diploidization of structurally retained duplicated R-genes.

## Discussion

### Diploidization following duplication as a major source of R-genes structural plasticity

Most of the investigated rosids (grape, *Arabidopsis*, soybean, poplar, and cacao) species experienced up to three WGD events, whereas the investigated grasses (rice, maize, sorghum, and *Brachypodium*) went through one shared ancestral WGD during their evolution, except for maize which experienced a recent extra-WGD 5 mya [66]. Biased erosion of duplicated gene redundancy between sister blocks has been characterized recently in plants defining

dominant and sensitive blocks [68,69]. In our current analysis, the identification of 23641 R-genes sequences in angiosperms established a higher R-genes conservation in grasses (on average 23.8%) compared to rosids (on average 6.6%) suggesting that successive rounds of WGDs act as a decay into R-genes conservation, as a primer source of R-gene plasticity. The evolutionary investigation of the characterized R-genes repertoire allowed the reconstruction of minimal ancestral pool of 465 and 150 founder R-genes respectively for the grasses and rosids.

Tandem duplication or clusterization played an important role in R-genes plasticity leading to structural variations such as CNV/PAV between species, which are thought to contribute to the reported tremendous R-genes diversity [85]. Special expansion of tandem duplications especially in sensitive chromosomes, as a rapid counterbalance flow of the duplicates deletion phenomenon, may have compensate R-genes loss in such chromosomal fragments as an expected consequence of the known diploidization process. R-genes clusters may have been shaped by classically proposed shuffling mechanisms such as replication slippage, segmental duplication *via* homologous/non-homologous unequal crossover, transposition *via* ectopic recombination/TE capture [86]. Such clusters may have been shaped by domain shuffling events [87], domain breakage and fusion so that R-domain combinations such as LRR-NBS-PK-TIR and LRR-NBS-PK-WRKY might be the result of local shuffling and recombination
<table><caption>**Table S12: R-genes cluster loci targeted by miRNAs in plants.** Cluster-loci, if several R-genes (at least two) are clustered distributing on the chromosome, this clustered location as one cluster loci; Cluster miRNA, At least one R-gene in Cluster loci can be targeted by miRNAs; %cluster-miRNA, percentage of cluster-loci regulated by miRNAs in the total cluster loci.</caption><thead><tr><th></th><th>Species</th><th>Cluster-loci #</th><th>Cluster-miRNA #</th><th>Cluster-miRNA %</th><th>Non-cluster #</th><th>Non-cluster-miRNA #</th><th>Non-cluster-miRNA %</th><th>P-value (Fisher's Exact Test)</th></tr></thead><tbody><tr><td rowspan="8">Eudicots</td><td>Arabidopsis</td><td>139</td><td>119</td><td>85.61</td><td>398</td><td>218</td><td>54.77</td><td>3.74E-03</td></tr><tr><td>Cacao</td><td>110</td><td>89</td><td>80.91</td><td>229</td><td>99</td><td>43.23</td><td>7.45E-06</td></tr><tr><td>Soybean</td><td>292</td><td>227</td><td>77.74</td><td>650</td><td>250</td><td>38.46</td><td>1.19E-09</td></tr><tr><td>Poplar</td><td>63</td><td>48</td><td>76.19</td><td>424</td><td>173</td><td>40.8</td><td>3.68E-03</td></tr><tr><td>Apple</td><td>460</td><td>327</td><td>71.09</td><td>550</td><td>240</td><td>43.64</td><td>3.85E-06</td></tr><tr><td>Lotus</td><td>47</td><td>33</td><td>70.21</td><td>537</td><td>177</td><td>32.96</td><td>2.98E-03</td></tr><tr><td>Papaya</td><td>48</td><td>26</td><td>54.17</td><td>150</td><td>22</td><td>14.67</td><td>9.75E-05</td></tr><tr><td>Strawberry</td><td>128</td><td>57</td><td>44.53</td><td>340</td><td>59</td><td>17.35</td><td>1.17E-05</td></tr><tr><td>Grape</td><td>102</td><td>44</td><td>43.14</td><td>195</td><td>22</td><td>11.28</td><td>1.80E-06</td></tr><tr><td rowspan="4">Monocots</td><td>Rice</td><td>231</td><td>173</td><td>74.89</td><td>436</td><td>183</td><td>41.97</td><td>1.68E-05</td></tr><tr><td>Sorghum</td><td>159</td><td>109</td><td>68.55</td><td>485</td><td>216</td><td>44.54</td><td>4.82E-03</td></tr><tr><td>Maize</td><td>31</td><td>18</td><td>58.06</td><td>769</td><td>233</td><td>30.3</td><td>4.91E-02</td></tr><tr><td>Brachypodium</td><td>132</td><td>69</td><td>52.27</td><td>315</td><td>96</td><td>30.48</td><td>4.89E-03</td></tr></tbody></table>
<figure><img src="image_7.png" /><figcaption>**Figure 4** R-genes/miRNAs interactome in plants. (A) Illustration of the percentage of R-genes targeted by miRNA in dicots species classified according the number of experienced WGDs (x-axis). The regression curve, correlation and associated *P*-value are mentioned. (B) Illustration of a micro-synteny locus between cacao (one region) and soybean (four duplicated regions) harboring R-genes targeted by miRNA (according to the number of sequence mismatches between miRNA and R-genes from 4 to 7 identified as miRNA target score and highlighted with a color code at the bottom). Grey bars represent non R-genes. (C) Illustration of the percentage of R-genes targeted by miRNA (y-axis) in dicots (D) and monocots (M) species classified according to the investigated R-domains (LRR, NBS, TIR, WRKY, Pkinase; x-axis).</figcaption></figure>

events. Altogether, R-gene clusterization, a center source of plasticity, triggered a serial of reshuffling events to make rapid copy variation leading to PAVs and CNVs between species, as a putative source for R-gene structural diversity. In the current analysis we observed up to 60% of R-genes organized in clusters in grasses and rosids. Genic and intergenic sequence repeats within R-clusters generated by duplication, transpositions and insertions provide a structural template that allows mis-pairing during recombination giving rise to unequal crossovers and interlocus gene conversions/rearrangements. The resulting R-domain combinations appeared not random with LRR as a 'glue' for domain association leading to new resistance gene isoforms in modern plant species.

### MicroRNA/R-gene interactome as a major source of R-genes functional diploidization

Recently, new evidences have been proposed regarding miRNAs regulating NBS-LRR in plants such as miR2109/ miR2118/miR1507 in Medicago, miR482/miR2118 in tomato, and miR6019/miR6020 in tobacco guiding the cleavage of transcript of NBS-LRRs, and then triggering the

secondary phased of siRNA production by RNA-dependent RNA polymerase [88-90]. Thus, MiRNAs may be involved in defense immunity in regulating R-genes expression level. Our *in silico* analysis suggests that miRNA may target preferentially duplicated R-genes either deriving from WGDs and more interestingly from local tandem duplications (*i.e.* clusters). This observation may suggest that the presence of redundant duplicated R-genes copies, when retained after diploidization, may require modification or specialization in expression/regulation through possibly miRNA interaction. Moreover, a specific R-domain affinity was observed for miRNA *in silico* interaction toward LRR/NBS/TIR, which may indicate to some extent a domain preference for R-gene/miRNA interaction. Overall, our data may suggest miRNAs as a dosage regulator playing a possible role in R-genes functional redundancy erosion following large or local duplication events. The preferential post-transcriptional regulation of duplicated R-genes by miRNA can be proposed as part of a functional diploidization process in response to duplications to maintain a perfect dosage balance regarding the product of R-genes duplicates.
<caption>**Figure S1:** R-genes and miRNA detection pipelines. (A) Method for R-genes characterization and mapping in plant genomes. (B) miRNAs target prediction for R-genes in plants. Calculation of CIP/CALP was performed according to Salse 2012. miRNA targets were predicted using Targetfinder software (see method section).</caption>

A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>
## Putative model of R-genes paleohistory in plants

Based on our analysis and previous studies, we proposed an hypothetical evolutionary model in Figure 5 illustrating the conclusion regarding the reported R-gene conservation/diversity, polyploidization, domain reshuffling as well

as microRNA/R-gene interactome. If duplicated R-genes are deleted between paralogous fragments after whole genome duplications following the general subgenome dominance hypothesis [68,69], they can be then considered as dosage-sensitive genes in returning to singleton status

<figure><img src="image_8.png" /></figure>
**Figure S2: R-domains distribution in plant genomes.** Three results of different methods in detecting R-gens including Annotation (Annot), Plant Resistance Gene database (PRGDB), and PFAM domain (Pfam) are as shown as colored bars. NR, total non-redundant R-genes content in integrating the three methods, LRR, NBS, TIR, LysM, RG contents are highlighted with the color code legend at the bottom of the figure.

<figure><img src="image_4.png" /></figure>
after WGDs and then are biased distributed on the modern pairs of duplicated chromosomes (Figure 5A). However, during evolution, if the deletion of R-genes in sensitive chromosome is compensated by high frequency of tandem duplication (clusterization) and/or transposition events, an equivalent number of R-genes between dominant and sensitive fragments is observed in modern plant genomes (Figure 5B). Despite the previous structural R-genes plasticity scenario, R-genes functional redundancy within clusters or in general between duplicated loci may be counterbalanced through microRNAs regulation with a specific affinity for LRR/NBS/TIR domains. Therefore, R-genes colinearity as well as subgenome dominance following WGDs has been eroded in the course of evolution. Finally R-genes clusterization may be considered as R-domain recombination hotspots, potential source of new domain combinations then possibly facilitating the neo-formation or neo-functionalization of R-genes isoforms (Figure 5C). Overall, when comparing modern plant species for their R-genes content, copy number variation (CNV), or Present/Absent variation (PAV) are generally observed (Figure 5D).

Conclusions

We reconstructed the R-genes paleohistory in plant un-
raveling duplications (either whole genome, small-scale
clusters or single-gene based) as the major source of struc-
tural (CNV, PAV, domain recombination) or even poten-
tially functional (enhanced miRNA regulation for R-gene
clusters and specific R-domains) plasticity that may have
promote the development a novel source of disease resis-
tance in the course of the evolution of the different inves-
tigated species. The conserved role of similar R-gene
families (especially TIR and NBS-LRR) in both plant and
animal defense systems suggest a common and ances-
tral origin. The current reconstruction of the ancestral
gene pool in angiosperm opens the perspective to deter-
mine the origin of innate immunity mechanism in
eukaryotes.

Methods

R-gene identification and mapping

R-genes were selected from monocots *Oryza sativa*, *Sorghum bicolor*, *Zea mays*, *Brachypodium distachyon*, and eudicots *Arabidopsis thaliana*, *Populus trichocarpa*, *Carica papaya*, *Glycine max*, *Lotus japonicas*, *Fragaria vesca*, and *Theobroma cacao*, on the basis of functional annotations available on Phytozome (http://www.phytozome.net/), Plant GDB (http://www.plantgdb.org/). *Vitis vinifera* and *Malus x domestica* R-genes annotation were retrieved from [59] and [60] supplementary data. The R-genes identification methods is illustrated in Additional file 1: Figure S1A. PFAM domain identification – Putative R-genes were investigated using profile Hidden Markov Model. Several PFAM profiles [64]

were used to extract putative R-gene proteins within the
13 genomes investigated: LRR: pf00560, pf07723, pf07725,
pf12799, pf01463, pf08263; NB-ARC: pf00931; TIR: pf01582;
LysM: pf01476; Pkinase: pf00069; WRKY: pf03106. PFAM
profiles were identified within genomes using the
hmmsearch algorithm (e-value cut: 1e-10) from HMMER3
(http://hmmer.janelia.org/; [91]). PRGdb – R-genes se-
quences from the Plant Resistance Gene database were
downloaded (http://prgdb.cbm.fvg.it/index.php, [65]). An-
notation, PFAM and PRGdb –based R-genes were aligned
against Soybean (46194 protein sequences), Cacao (27814
protein sequences), Strawberry (34809 protein sequences),
Lotus (15470 protein sequences), Papaya (19205 protein
sequences), Poplar (30260 protein sequences), Apple
(58979 protein sequences), *Brachypodium*, (32255 pro-
tein sequences) Sorghum (36338 protein sequences),
and Maize (53764 protein sequences) genome data
using BLASTP (PFAM and Annotation R-gene se-
quences) and BLASTX (PRGdb sequences). BLAST re-
sults were parsed using CIP (Cumulative Identity
Percentage) and CALP (Cumulative Alignment Length
Percentage) parameters (70% as minimum threshold)
delivering a non-redundant list of R-genes for each spe-
cies [44].

Orthologs/Paralogs identification and synteny
relationships

Orthologous and paralogous R-genes were identified align-
ing Rice RefBank against *Brachypodium*, Sorghum and
Maize using BLASTALL. BLASTP results are parsed with
CIP and CALP parameters set to 60% and 70% as mini-
mum threshold for ortholog identification and 60% and
70% as minimum threshold for paralog identification as de-
scribed in [44]. Ancestral relationship between monocot
species were represented as concentric circles with the
visualization tool Circos [92]. Relationship between rosids
species were investigated with the same protocol. Grape
RefBank was aligned against Soybean, Cacao, Strawberry,
Lotus, Papaya, Poplar and Apple genome data.
BLAST results were parsed with the same parameters
described previously. The synteny relations at the
chromosome levels were considered using public syn-
teny data available for both Monocotyledones and
Eudicotyledones [66].

R-genes not located in the syntenic region were BLAST
aligned against the total R-genes content. The gene pairs ex-
cluding self matches (CIP > = 70%, CIAP > = 70%) were
considered as single-gene duplication and used to the fur-
ther analysis. Then we selected 40 flanking genes windows
surrounding R-genes pairs. If flanking pairs with E-value
<= e-10 are observed these paired R-genes were then
considered as part of a segmental duplication, otherwise,
as single-gene duplication.
**Figure S3: R-genes family distribution in plant genomes.** The color code illustrates the R-gene families PTI/ETI/R-combination/R-pathway characterized for the 13 plant genomes including monocots (rice, *Brachypodium*, sorghum, and maize) and eudicots (*Arabidopsis*, Grape, Cacao, Papaya, Strawberry, Poplar, Lotus, Apple, and Soybean).

<figure><img src="image_5.png" /></figure>

<table><tr><td>PTI</td><td>ETI</td><td>R-combination</td><td>R-pathway</td></tr></table>
## Permutation-test for R-genes partitioning between duplicated blocks

In the absence of any biased retention/deletion of ancestral R-genes content (N) after WGD (null hypothesis), the post-duplication R-genes content is 2 N = N + N. After million years of evolution and associated shuffling, n1 and n2 R-genes are observed in modern duplicated chromosomes. We simulated the random deletion of R-genes 1000 times (number of deleted R-gene is equal with (N - n1) and (N - n2) in the two duplicated chromosomes respectively), and derived two sample datasets (Sample1: X₁, X₂, X₃,....X₁₀₀; Sample: Y₁, Y₂, Y₃,....Y₁₀₀) corresponding to the random deletion of R-genes between blocks. We performed Z-tests (see formula below) to test significant differences in the distribution of retained R-genes between the two duplicated chromosome pairs (null hypothesis rejected).

$$Z = \frac{X-u}{\frac{\sigma}{\sqrt{n}}} \left( u = \frac{\sum_{i=0}^{1000} (X_i - Y_i)}{n} \right); \\ X = n1-n2; n = 1000; \sigma^2, \text{variance}$$

## MiRNA identification associated with R-genes as targets

Mature miRNAs dataset from miRBase (http://www.mirbase.org/; Release 18) was used to predict R-genes as targets in the investigated plant genomes Targetfinder algorithm (http://carringtonlab.org/resources/targetfinder/) with score < 4 [84]. To reduce the false positive, secondary structures of the identified mature miRNA was validated using MiReNA software [93]. R-genes targeted by miRNA with validated secondary structure and with a mismatch score < 4, are considered as *in silico* targets of miRNA. The detailed pipeline is illustrated in Additional file 1: Figure S1B.

## Additional files

**Additional file 1: Table S1.** R-genes conservation in plants. **Table S2.** R-genes domains/family diversity in plants. **Table S3.** Number of R-genes in the ancestral duplicated chromosomes in grasses. **Table S4.** Number of R-genes in the recent duplicated maize chromosomes. **Table S5.** Number of R-genes clusters in ancient duplicated grass chromosomes.

**Table S6.** Number of R-genes clusters in recent duplicated maize chromosomes. **Table S7.** R-genes clusters distribution in plants. **Table S8.** R-genes duplication frequency in maize. **Table S9.** R-genes duplication frequency in maize compared to other grasses. **Table S10.** R-genes targeted by miRNAs in plants. **Table S11.** R-domains targeted by miRNAs in eudicots.

**Table S12.** R-genes cluster loci targeted by miRNAs in plants. **Figure S1.** R-genes and miRNA detection pipelines. **Figure S2.** R-domains distribution in plant genomes. **Figure S3.** R-genes family distribution in plant genomes.

**Figure S4.** R-genes paleohistorical evolution in eudicots. **Figure S5.** Evolutionary scenario of R-genes families in monocots. **Figure S6.** R-genes distribution and content in clusters. **Figure S7.** R-domains combination in clusters. **Figure S8.** R-gene clusters targeted by miRNAs in plants.

**Additional file 2: Dataset S1.** R-gene repertoire and associated miRNAs in plants. The table provides the catalog of R-genes characterized in grasses including rice (OS), *Brachypodium* (BD), sorghum (SB), and maize (ZM), and rosids including grape (WV), *Arabidopsis* (AT), papaya (CP), Cacao (TC), Soybean (GM), Lotus (LJ), Medicago (MD), Strawberry (FV), and Poplar (PT).

**Additional file 3: Dataset S2.** Ancestral R-gene content in grass ancestor. The table provides the catalog of R-genes characterized in the 5 ancestral chromosomes of grasses with associated R-domains and modern R-genes representatives characterized on rice, *Brachypodium*, sorghum and maize duplicated fragments.

**Additional file 4: Dataset S3.** Duplicated R-genes repertoire in grasses. The table provides the catalog of non-syntenic duplicated R-genes characterized in rice (OS), *Brachypodium* (BD), sorghum (SB), and maize (ZM).

## Competing interests

The authors (RZ, FM, CP, TL and JS) declare they have no competing interests as part of the same research unit (INRA/UBP UMR 1095 GDEC 'Génétique, Diversité et Ecophysiologie des Céréales', 5 chemin de Beaulieu, 63100 Clermont-Ferrand, France) when the study performed.

## Authors' contributions

RZ, FM, and CP performed the analyses. RZ, TL and JS wrote the manuscript. All authors read and approved the final manuscripts.

## Acknowledgements

This work has been supported by grants from the Agence Nationale de la Recherche (program ANR Blanc-PAGE, ref: ANR-2011-BSV6-00801).

## Author details

¹INRA/UBP UMR 1095 GDEC 'Génétique, Diversité et Ecophysiologie des Céréales', 5 chemin de Beaulieu, 63100 Clermont-Ferrand, France. ²Institute of Crop Sciences, Shandong Academy of Agricultural Sciences, Jinan 250100, China.

Received: 5 September 2013 Accepted: 25 February 2014

Published: 12 March 2014

## References

1. Glazebrook J: Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. *Annu Rev Phytopathol* 2005, 43:205-227.

2. Pallas V, Garcia JA: How do plant viruses induce disease? Interactions and interference with host components. *J Gen Virol* 2011, 92(Pt 12):2691-2705.

3. Soriano IR, Riley IT, Potter MJ, Bowers WS: Phytoecysteroids: a novel defense against plant-parasitic nematodes. *J Chem Ecol* 2004, 30(10):1885-1899.

4. Choy A, Roy CR: Autophagy and bacterial infection: an evolving arms race. *Trends Microbiol* 2013, 21(9):451-456.

5. Chisholm ST, Coaker G, Day B, Staskawicz BJ: Host-microbe interactions: shaping the evolution of the plant immune response. *Cell* 2006, 124(4):803-814.

6. Medzhitov R, Janeway CA Jr: Innate immunity: the virtues of a nonclonal system of recognition. *Cell* 1997, 91(3):295-298.

7. Nurnberger T, Brunner F: Innate immunity in plants and animals: emerging parallels between the recognition of general elicitors and pathogen-associated molecular patterns. *Curr Opin Plant Biol* 2002, 5(4):318-324.

8. Mur LAJ, Kenton P, Lloyd AJ, Ougham H, Prats E: The hypersensitive response; the centenary is upon us but how much do we know? *J Exp Bot* 2008, 59(3):20.

9. Hammond-Kosack KE, Parker JE: Deciphering plant-pathogen communication: fresh perspectives for molecular resistance breeding. *Curr Opin Biotechnol* 2003, 14(2):177-193.

10. Glowacki S, Macioszek VK, Kononowicz AK: R proteins as fundamentals of plant innate immunity. *Cell Mol Biol Lett* 2011, 16(1):1-24.

11. Qiu D, Xiao J, Ding X, Xiong M, Cai M, Cao Y, Li X, Xu C, Wang S: OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. *Mol Plant Microbe Interact* 2007, 20(5):492-499.

12. Shimono M, Koga H, Akagi A, Hayashi N, Goto S, Sawada M, Kurihara T, Matsushita A, Sugano S, Jiang CJ, Kaku H, Inoue H, Takatsuji H: Rice WRKY45 plays important roles in fungal and bacterial disease resistance. *Mol Plant Pathol* 2012, 13(1):83-94.

13. Spaink HP: Specific recognition of bacteria by plant LysM domain receptor kinases. *Trends Microbiol* 2004, 12(5):201-204.

14. Buist G, Steen A, Kok J, Kuipers OP: LysM, a widely distributed protein motif for binding to (peptido)glycans. *Mol Microbiol* 2008, 68(4):838-847.

15. Witte CP, Keinath N, Dubiella U, Demouliere R, Seal A, Romeis T: Tobacco calcium-dependent protein kinases are differentially phosphorylated in vivo as part of a kinase cascade that regulates stress response. *J Biol Chem* 2010, 285(13):9740-9748.
<figure><img src="image_6.png" /><figcaption>**Figure S4: R-genes paleohistorical evolution in eudicots.** The modern genome structures of eudicots are depicted with a seven color code that illuminate their relationships with n = 7 AGK (top) in respectively. The percentages of resistant domains (reference as color code legend at the bottom) are shown with circular distributions for the nine eudicot genomes (bottom) investigated.</figcaption></figure>
16. Kurusu T, Hamada J, Nokajima H, Kitagawa Y, Kiyoduka M, Takahashi A, Hanamata S, Ohno R, Hayashi T, Okada K, Koga J, Hirochika H, Yamane H, Kuchitsu K: Regulation of microbe-associated molecular pattern-induced hypersensitive cell death, phytoalexin production, and defense gene expression by calcineurin B-like protein-interacting protein kinases, OsCIPK14/15, in rice cultured cells. *Plant Physiol* 2010, **153**(2):678–692.

17. Bent AF: Plant disease resistance genes: function meets structure. *Plant Cell* 1996, **8**(10):1757–1771.

18. Wang J, Tan S, Zhang L, Li P, Tian D: Co-variation among major classes of LRR-encoding genes in two pairs of plant species. *J Mol Evol* 2011, **72**(5-6):498–509.

19. Panstruga R, Parker JE, Schulze-Lefert P: Snapshot: plant immune response pathways. *Cell* 2009, **136**(5):978, e971-973.

20. Yue JX, Meyers BC, Chen JQ, Tian D, Yang S: Tracing the origin and evolutionary history of plant nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes. *New Phytol* 2012, **193**(4):1049–1063.

21. Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S, Sobral BW, Young ND: Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. *Plant J* 1999, **20**(3):317–332.

22. Akita M, Valkonen JP: A novel gene family in moss (Physcomitrella patens) shows sequence homology and a phylogenetic relationship with the TIR-NBS class of plant disease resistance genes. *J Mol Evol* 2002, **55**(5):595–605.

23. Bertin J, Nir WJ, Fischer CM, Tayber D, Errada PR, Grant JR, Keity JJ, Gosselin ML, Robison KE, Wong GH, Glucksmann MA, DiStefano PS: Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kappaB. *J Biol Chem* 1999, **274**(19):12955–12958.

24. Meyers BC, Morgante M, Michelmore RW: TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsis and other plant genomes. *Plant J* 2002, **32**(1):77–92.

25. Liu JJ, Ekramoddoullah AK: Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine (Pinus monticola Dougl. ex. D. Don). *Mol Genet Genomics* 2003, **270**(5):432–441.

26. Girardin SE, Sansonetti PJ, Philpott DJ: Intracellular vs extracellular recognition of pathogens–common concepts in mammals and flies. *Trends Microbiol* 2002, **10**(4):193–199.

27. Tarr DE, Alexander HM: TIR-NBS-LRR genes are rare in monocots: evidence from diverse monocot orders. *BMC Res Notes* 2009, **2**:197.

28. Bai J, Pennill LA, Ning J, Lee SW, Ramalingam J, Webb CA, Zhao B, Sun Q, Nelson JC, Leach JE, Hulbert SH: Diversity in nucleotide binding site-leucine-rich repeat genes in cereals. *Genome Res* 2002, **12**(12):1871–1884.

29. Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW: Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. *Plant Cell* 2003, **15**(4):809–834.

30. Yang S, Zhang X, Yue JX, Tian D, Chen JQ: Recent duplications dominate NBS-encoding gene expansion in two woody species. *Mol Genet Genomics* 2008, **280**(3):187–198.

31. Porter BW, Paidi M, Ming R, Alam M, Nishijima WT, Zhu YJ: Genome-wide analysis of Carica papaya reveals a small NBS resistance gene family. *Mol Genet Genomics* 2009, **281**(6):609–626.

32. Leister D: Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance gene. *Trends Genet* 2004, **20**(3):116–122.

33. Luo S, Zhang Y, Hu Q, Chen J, Li K, Lu C, Liu H, Wang W, Kuang H: Dynamic nucleotide-binding site and leucine-rich repeat-encoding genes in the grass family. *Plant Physiol* 2012, **159**(1):197–210.

34. Guo YL, Fitz J, Schneeberger K, Ossowski S, Cao J, Weigel D: Genome-wide comparison of nucleotide-binding site-leucine-rich repeat-encoding genes in Arabidopsis. *Plant Physiol* 2011, **157**(2):757–769.

35. Shen J, Araki H, Chen L, Chen JQ, Tian D: Unique evolutionary mechanism in R-genes under the presence/absence polymorphism in Arabidopsis thaliana. *Genetics* 2006, **172**(2):1243–1250.

36. Yang S, Feng Z, Zhang X, Jiang K, Jin X, Hang Y, Chen JQ, Tian D: Genome-wide investigation on the genetic variations of rice disease resistance genes. *Plant Mol Biol* 2006, **62**(1–2):181–193.

37. Leister D, Kurth J, Laurie DA, Yano M, Sasaki T, Devos K, Graner A, Schulze-Lefert P: Rapid reorganization of resistance gene homologues in cereal genomes. *Proc Natl Acad Sci U S A* 1998, **95**(1):370–375.

38. Meyers BC, Kaushik S, Nandety RS: Evolving disease resistance genes. *Curr Opin Plant Biol* 2005, **8**(2):129–134.

39. Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H,

Wang X, Wicker T, Bharti AK, Chapman J, Feltus FA, Gowik U, Grigoriev IV, Lyons E, Maher CA, Martins M, Narechania A, Otillar RP, Penning BW, Salamov AA, Wang Y, Zhang L, Carpita NC, et al: The Sorghum bicolor genome and the diversification of grasses. *Nature* 2009, **457**(7229):551–556.

40. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, Du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, et al: The B73 maize genome: complexity, diversity, and dynamics. *Science* 2009, **326**(5956):1112–1115.

41. International Rice Genome Sequencing P: The map-based sequence of the rice genome. *Nature* 2005, **436**(7052):793–800.

42. International Brachypodium I: Genome sequencing and analysis of the model grass Brachypodium distachyon. *Nature* 2010, **463**(7282):763–768.

43. Salse J, Abrouk M, Bolot S, Guilhot N, Courcelle E, Faraut T, Waugh R, Close TJ, Messing J, Feuillet C: Reconstruction of monocotelydoneous proto-chromosomes reveals faster evolution in plants than in animals. *Proc Natl Acad Sci U S A* 2009, **106**(35):14908–14913.

44. Salse J, Abrouk M, Murat F, Quraishi UM, Feuillet C: Improved criteria and comparative genomics tool provide new insights into grass paleogenomics. *Brief Bioinform* 2009, **10**(6):619–630.

45. Bolot S, Abrouk M, Masood-Quraishi U, Stein N, Messing J, Feuillet C, Salse J: The 'inner circle' of the cereal genomes. *Curr Opin Plant Biol* 2009, **12**(2):119–125.

46. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choïsne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyère C, Billault A, Segurens B, Gouvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyta. *Nature* 2007, **449**(7161):463–467.

47. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, Schein J, Sterck L, Aerts A, Bhalerao RR, Bhalerao RP, Blaudez D, Boerjan W, Brun A, Brunner A, Busov V, Campbell M, Carlson J, Chalot M, Chapman J, Chen GL, Cooper D, Coutinho PM, Couturier J, Covert S, Cronk Q, et al: The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). *Science* 2006, **313**(5793):1596–1604.

48. Arabidopsis Genome I: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. *Nature* 2000, **408**(6814):796–815.

49. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, et al: Genome sequence of the palaeopolyploid soybean. *Nature* 2010, **463**(7278):178–183.

50. Argout X, Salse J, Aury JM, Guiltinan MJ, Droc G, Gouzy J, Allegre M, Chaparro C, Legavre T, Maximova SN, Abrouk M, Murat F, Fouet O, Poulain J, Ruiz M, Roguet Y, Rodier-Goud M, Barbosa-Neto JF, Sabot F, Kudra D, Ammiraju JS, Schuster SC, Carlson JE, Sallet E, Schiex T, Dievart A, Kramer M, Gelley L, Shi Z, Bérard A, et al: The genome of Theobroma cacao. *Nat Genet* 2011, **43**(2):101–108.

51. Abrouk M, Murat F, Pont C, Messing J, Jackson S, Faraut T, Tannier E, Plomion C, Cooke R, Feuillet C, Salse J: Palaeogenomics of plants: synteny-based modelling of extinct ancestors. *Trends Plant Sci* 2010, **15**(9):479–487.

52. Paterson AH, Bowers JE, Chapman BA: Ancient polyploidization predating divergence of the cereals, and its consequences for comparative genomics. *Proc Natl Acad Sci U S A* 2004, **101**(26):9903–9908.

53. Tang H, Bowers JE, Wang X, Ming R, Alam M, Paterson AH: Synteny and collinearity in plant genomes. *Science* 2008, **320**(5875):486–488.

54. Tang H, Wang X, Bowers JE, Ming R, Alam M, Paterson AH: Unraveling ancient hexaploidy through multiply-aligned angiosperm gene maps. *Genome Res* 2008, **18**(12):1944–1954.

55. Van de Peer Y, Fawcett JA, Proost S, Sterck L, Vandepoele K: The flowering world: a tale of duplications. *Trends Plant Sci* 2009, **14**(12):680–688.

56. Bowers JE, Chapman BA, Rong J, Paterson AH: Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. *Nature* 2003, **422**(6930):433–438.

57. Lawton-Raeh A: Evolutionary dynamics of duplicated genes in plants. *Mol Phylogenet Evol* 2003, **29**(3):396–409.

58. Blanc G, Wolfe KH: Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. *Plant Cell* 2004, **16**(7):1667–1678.
<figure><img src="image_7.png" /><figcaption>**Figure S5:** Evolutionary scenario of R-gene families in monocots. The modern grass genome structures (bottom) are depicted with a five-color code that illuminates their relationship with the *n* = 5 (A5, A7, A11, A8, A4) and *n*=12 (A1 to A12) ancestors (top), according to Murat et al. . The characterized R-genes are illustrated as vertical bars on the chromosomes of modern and ancestral genomes. The percentages of R-gene classes (LRR, NBS, TIR, LysM, RG, highlighted with the color code legend at the bottom) are shown with circular distributions for the four monocot genomes (bottom), the rice/*Brachypodium* and sorghum/maize ancestral genome intermediates (center), as well as for the ancestral genomes (top). Statistically enriched and impoverished R-gene families (PTI/ETI/R-combination/R-pathway depicted with a four-color code) are illustrated with red and blue stars on the circular distributions, respectively.</figcaption></figure>
59. Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss D, Pindo M, Fitzgerald LM, Vezzulli S, Reid J, Malacarne G, Iliev D, Coppola G, Wardell B, Micheletti D, Macalma T, Facci M, Mitchell JT, Perazzolli M, Eldredge G, Gatto P, Oyzerski R, Moretto M, Gutin N, Stefanini M, Chen Y, Segala C, Davenport C, Dematté L, Mraz A, et al: A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. *PLoS One* 2007, 2(12):e1326.

60. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, et al: The genome of the domesticated apple (Malus x domestica Borkh). *Nat Genet* 2010, 42(10):833-839.

61. Sato S, Nakamura Y, Kaneko T, Asamizu E, Kato T, Nakao M, Sasamoto S, Watanabe A, Ono A, Kawashima K, Fujishiro T, Katoh M, Kohara M, Kishida Y, Minami C, Nakayama S, Nakazaki N, Shimizu Y, Shinpo S, Takahashi C, Wada T, Yamada M, Ohmido N, Hayashi M, Fukui K, Baba T, Nakamichi T, Mori H, Tabata S: *Genome structure of the legume, Lotus japonicus*. *DNA Res* 2008, 15(4):227-239.

62. Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, Burns P, Davis TM, Slovin JP, Bassil N, Hellens RP, Evans C, Harkins T, Kodira C, Desany B, Crasta OR, Jensen RV, Allan AC, Michael TP, Setubal JC, Celton JM, Rees DJ, Williams KP, Holt SH, Ruiz Rojas JJ, Chatterjee M, et al: The genome of woodland strawberry (Fragaria vesca). *Nat Genet* 2011, 43(2):109-116.

63. Ming R, Hou S, Feng Y, Yu Q, Dionne-Laporte A, Saw JH, Senin P, Wang W, Ly BV, Lewis KL, Salzberg SL, Feng L, Jones MR, Skelton RL, Murray JE, Chen C, Qian W, Shen J, Du P, Eustice M, Tong E, Tang H, Lyons E, Paull RE, Michael TP, Wall K, Rice DW, Albert H, Wang ML, Zhu YJ: The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). *Nature* 2008, 452(7190):991-996.

64. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M: The Pfam protein families database. *Nucleic Acids Res* 2014, 42 part 2(Database issue):D222-D230.

65. Sanseverino W, Roma G, De Simone M, Faino L, Melito S, Stupka E, Frusciante L, Ercolano MR: PRGdb: a bioinformatics platform for plant resistance gene analysis. *Nucleic Acids Res* 2010, 38(Database issue):D814-D821.

66. Salse J: In silico archeogenomics unveils modern plant genome organisation, regulation and evolution. *Curr Opin Plant Biol* 2012, 15(2):122-130.

67. Murat F, Xu JH, Tannier E, Abrouk M, Guilhot N, Pont C, Messing J, Salse J: Ancestral grass karyotype reconstruction unravels new mechanisms of genome shuffling as a source of plant evolution. *Genome Res* 2010, 20(11):1545-1557.

68. Abrouk M, Zhang R, Murat F, Li A, Pont C, Mao L, Salse J: Grass microRNA gene paleohistory unveils new insights into gene dosage balance in subgenome partitioning after whole-genome duplication. *Plant Cell* 2012, 24(5):1776-1792.

69. Schnable JC, Freeling M, Lyons E: Genome-wide analysis of syntenic gene deletion in the grasses. *Genome Biol Evol* 2012, 4(3):265-277.

70. Thomas BC, Pedersen B, Freeling M: Following tetraploidy in an Arabidopsis ancestor, genes were removed preferentially from one homeolog leaving clusters enriched in dose-sensitive genes. *Genome Res* 2006, 16(7):934-946.

71. Sankoff D, Zheng C, Zhu Q: The collapse of gene complement following whole genome duplication. *BMC Genomics* 2010, 11:313.

72. Xiong Y, Liu T, Tian C, Sun S, Li J, Chen M: Transcription factors in rice: a genome-wide comparative analysis between monocots and eudicots. *Plant Mol Biol* 2005, 59(1):191-203.

73. Freeling M: Bias in plant gene content following different sorts of duplication: tandem, whole-genome, segmental, or by transposition. *Annu Rev Plant Biol* 2009, 60:433-453.

74. Richly E, Kurth J, Leister D: Mode of amplification and reorganization of resistance genes during recent Arabidopsis thaliana evolution. *Mol Biol Evol* 2002, 19(1):76-84.

75. Xu Z, Wang H: LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. *Nucleic Acids Res* 2007, 35(Web Server issue):W265-W268.

76. Wicker T, Buchmann JP, Keller B: Patching gaps in plant genomes results in gene movement and erosion of colinearity. *Genome Res* 2010, 20(9):1229-1237.

77. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP: MicroRNAs in plants. *Genes Dev* 2002, 16(13):1616-1626.

78. Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP: Vertebrate microRNA genes. *Science* 2003, 299(5612):1540.

79. Bagga S, Pasquinelli AE: Identification and analysis of microRNAs. *Genet Eng* 2006, 27:1-20.

80. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD: A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. *Science* 2006, 312(5772):436-439.

81. Li Y, Zhang Q, Zhang J, Wu L, Qi Y, Zhou JM: Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity. *Plant Physiol* 2010, 152(4):2222-2231.

82. Du P, Wu J, Zhang J, Zhao S, Zheng H, Gao G, Wei L, Li Y: Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors. *PLoS Pathog* 2011, 7(8):e1002176.

83. Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A Jr, Zhu JK, Staskawicz BJ, Jin H: A pathogen-inducible endogenous siRNA in plant immunity. *Proc Natl Acad Sci U S A* 2006, 103(47):18002-18007.

84. Allen E, Xie Z, Gustafson AM, Carrington JC: microRNA-directed phasing during trans-acting siRNA biogenesis in plants. *Cell* 2005, 121(2):207-221.

85. McHale LK, Haun WJ, Xu WW, Bhaskar PB, Anderson JE, Hyten DL, Gerhardt DJ, Jeddeloh JA, Stupar RM: Structural variants in the soybean genome localize to clusters of biotic stress-response genes. *Plant Physiol* 2012, 159(4):1295-1308.

86. Chantret N, Salse J, Sabot F, Rahman S, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P, Joudrier P, Gautier MF, Cattolico L, Beckert M, Aubourg S, Weissenbach J, Caboche M, Bernard M, Leroy P, Chalhoub B: Molecular basis of evolutionary events that shaped the hardness locus in diploid and polyploid wheat species (Triticum and Aegilops). *Plant Cell* 2005, 17(4):1033-1045.

87. Sun X, Wang GL: Genome-wide identification, characterization and phylogenetic analysis of the rice LRR-kinases. *PLoS One* 2011, 6(3):e16079.

88. Zhai J, Jeong DH, De Paoli E, Park S, Rosen BD, Li Y, González AJ, Yan Z, Kitto SL, Grusak MA, Jackson SA, Stacey G, Cook DR, Green PJ, Sherrier DJ, Meyers BC: MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. *Genes Dev* 2011, 25(23):2540-2553.

89. Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM, Tung J, Sun H, Kumar P, Baker B: MicroRNA regulation of plant innate immune receptors. *Proc Natl Acad Sci U S A* 2012, 109(5):1790-1795.

90. Eckardt NA: A microRNA cascade in plant defense. *Plant Cell* 2012, 24(3):840.

91. Finn RD, Clements J, Eddy SR: HMMER web server: interactive sequence similarity searching. *Nucleic Acids Res* 2011, 39(Web Server issue):W29-W37.

92. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA: Circos: an information aesthetic for comparative genomics. *Genome Res* 2009, 19(9):1639-1645.

93. Mathelier A, Carbone A: MiRerna: finding microRNAs with high accuracy and no learning at genome scale and from deep sequencing data. *Bioinformatics* 2010, 26(18):2226-2234.

doi:10.1186/1471-2164-15-187

Cite this article as: Zhang et al.: Paleo-evolutionary plasticity of plant disease resistance genes. *BMC Genomics* 2014 15:187.

Submit your next manuscript to BioMed Central and take full advantage of:

* Convenient online submission

* Thorough peer review

* No space constraints or color figure charges

* Immediate publication on acceptance

* Inclusion in PubMed, CAS, Scopus and Google Scholar

* Research which is freely available for redistribution

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central
**Figure S6: R-genes distribution and content in clusters.** The X-axis represents gene number of R-genes in clusters; the Y-axis represents the percentage of observed clusters with OS for rice BD for *Brachypodium*, SB for sorghum and ZM for maize.

<figure><img src="image_8.png" /></figure>
**Figure S7: R-domains combination in clusters.** (A) Illustration of the different domain (color code) combinations observed in the characterized R-gene clusters. (B) Domain distribution in the combined R-genes. Y axis represents the percentage of domains and X axis represents the different domains. (C) Using LRR domain as a reference, the distribution of the other domains combined with LRR are shown for O, B, S, Z, V, A, C, F, G, L, M, P, T representing rice, *Brachypodium*, Sorghum, Maize, Grape, *Arabidopsis*, Papaya, Strawberry, Soybean, Lotus, Apple, Poplar and Cacao respectively.

<figure><img src="image_9.png" /></figure>
**Figure S8: R-genes clusters targeted by miRNAs in plants.** X-axis represents the investigated species including eudicots (*Arabidopsis*/*AT*, Cacao/*TC*, Soybean/*GM*, Poplar/*PT*, Apple/*MD*, Lotus/*LJ*, Papaya/*CP*, Strawberry/*FV*, and grape/*VV*), and monocots (Rice/*OS*, Sorghum/*SB*, Maize/*ZM*, and *Brachypodium/*BD*); Y-axis represents the percentage of R-genes loci regulated by miRNAs (either cluster or non cluster).

<figure><img src="image_10.png" /></figure>

**Eudicots:** *AT*, *Arabidopsis*; *TC*, Cacao; *GM*, Soybean; *PT*, Poplar; *MD*, Apple; *LJ*, Lotus; *CP*, Papaya; *FV*, Strawberry; *VV*, grape/VV,

**Monocots:** *OS*, Rice; *SB*, Sorghum; *ZM*, Maize; *BD*, *Brachypodium*