Comparative analysis of Phytophthora genomes reveals
oomycete pathogenesis in crops

Rui-Fang Gao, Jie-Yu Wang, Ke-Wei Liu, Kouki Yoshida, Yu-Yun Hsiao, Yi-Xiang Shi, Kun-Chan Tsai, You-Yi Chen, Nobutaka Mitsuda, Chieh-Kai Liang, et al.

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

► To cite this version:

Rui-Fang Gao, Jie-Yu Wang, Ke-Wei Liu, Kouki Yoshida, Yu-Yun Hsiao, et al. Comparative analysis of Phytophthora genomes reveals oomycete pathogenesis in crops. Heliyon, 2021, 7 (2), pp.e06317. 10.1016/j.heliyon.2021.e06317. hal-04003903

HAL Id: hal-04003903

https://hal.inrae.fr/hal-04003903

Submitted on 24 Feb 2023

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire **HAL**, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Comparative analysis of *Phytophthora* genomes reveals oomycete
pathogenesis in crops

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

Check for
updates

Rui-Fang Gao<sup>a,b,1</sup>, Jie-Yu Wang<sup>c,d,1</sup>, Ke-Wei Liu<sup>e,f,g,1</sup>, Kouki Yoshida<sup>h,1</sup>, Yu-Yun Hsiao<sup>i,1</sup>, Yi-Xiang Shi<sup>j</sup>, Kun-Chan Tsai<sup>k</sup>, You-Yi Chen<sup>l</sup>, Nobutaka Mitsuda<sup>m</sup>, Chieh-Kai Liang<sup>l</sup>, Zhi-Wen Wang<sup>n</sup>, Ying Wang<sup>a,b</sup>, Di-Yang Zhang<sup>c</sup>, Laiqiang Huang<sup>e,f</sup>, Xiang Zhao<sup>n</sup>, Wen-Ying Zhong<sup>n</sup>, Ying-Hui Cheng<sup>o</sup>, Zi-De Jiang<sup>p</sup>, Ming-He Li<sup>c</sup>, Wei-Hong Sun<sup>c</sup>, Xia Yu<sup>c</sup>, Wenqi Hu<sup>c</sup>, Zhuang Zhou<sup>c,q</sup>, Xiao-Fan Zhou<sup>p</sup>, Chuan-Ming Yeh<sup>m,r,s,**</sup>, Kazutaka Katoh<sup>t,***</sup>, Wen-Chieh Tsai<sup>i,l,u,****</sup>, Zhong-Jian Liu<sup>c,f,q,v,*</sup>, Francis Martin<sup>w,*****</sup>, Gui-Ming Zhang<sup>a,b,******</sup>

<footnote><sup>a</sup> Animal & Plant Inspection and Quarantine Technology Center of Shenzhen Customs District P.R. China, Shenzhen 518045, China</footnote>

<footnote>b Shenzhen Key Laboratory for Research & Development on Detection Technology of Alien Pests, Shenzhen Academy of Inspection and Quarantine, Shenzhen 518045, China</footnote>

<footnote>c Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization at College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou 350002, China</footnote>

<footnote>d Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China</footnote>

<footnote>e School of Life Sciences, Tsinghua University, Beijing 100084, China</footnote>

<footnote>f Center for Biotechnology and Biomedicine, Shenzhen Key Laboratory of Gene and Antibody Therapy State Key Laboratory of Health Sciences and Technology (prep), Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China</footnote>

<footnote>g Center for Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute (TBSI), Shenzhen 518055, China</footnote>

<footnote>h Technology Center, Taisei Corporation, Nase-cho 344-1, Totsuka-ku, Yokohama, Kanagawa 245-0051, Japan</footnote>

<footnote>i Orchid Research and Development Center, National Cheng Kung University, Tainan 701, Taiwan</footnote>

<footnote>j Shanghai Major Bio-pharm Technology Co., Ltd., Shanghai 201203, China</footnote>

<footnote>k Reber Genetics Co., Ltd., Taipei 10685, Taiwan</footnote>

<footnote>l Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan</footnote>

<footnote>m Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan</footnote>

<footnote>n PubBio-Tech Services Corporation, Wuhan 430070, China</footnote>

<footnote>o Fairylake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen 518004, China</footnote>

<footnote>P College of Agriculture, South China Agricultural University, Guangzhou 510640, China</footnote>

<footnote>q Zhejiang Institute of Subtropical Crops, Zhejiang Academy of Agricultural Sciences, Wenzhou 325005, China</footnote>

<footnote>r Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan</footnote>

<footnote>S Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan</footnote>

<footnote>t Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan</footnote>

<footnote>u Institute of Tropical Plant Sciences and Microbiology, National Cheng Kung University, Tainan 701, Taiwan</footnote>

<footnote>v Institute of Vegetable and Flowers, Shandong Academy of Agricultural Sciences, 250100, Jinan, China</footnote>

<footnote>w Institut National de la Recherche Agronomique, UMR Interactions Arbres/Microorganismes, Centre INRA Grand Est-Nancy, Université de Lorraine, 54280 Champenoux, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>*** Corresponding author.</footnote>

<footnote>**** Corresponding author.</footnote>

<footnote>***** Corresponding author.</footnote>

<footnote>****** Corresponding author.</footnote>
ARTICLE INFO

Keywords:

Phytophthora

Genome

Phylogenetic

Pathogenicity

Horizontally gene transfer

A B S T R A C T

The oomycete genus *Phytophthora* includes devastating plant pathogens that are found in almost all ecosystems. We sequenced the genomes of two quarantined *Phytophthora* species–*P. fragariae* and *P. rubi*. Comparing these *Phytophthora* species and related genera allowed reconstruction of the phylogenetic relationships within the genus *Phytophthora* and revealed *Phytophthora* genomic features associated with infection and pathogenicity. We found that several hundred *Phytophthora* genes are putatively inherited from red algae, but *Phytophthora* does not have vestigial plastids originating from phototrophs. The horizontally-transferred *Phytophthora* genes are from abundant transposon activities that “transmit” exogenous genes to *Phytophthora* species and thus bring about the gene recombination possibility. Several expansion events of *Phytophthora* gene families associated with cell wall biogenesis can be used as mutational targets to elucidate gene function in pathogenic interactions with host plants. This work enhanced the understanding of *Phytophthora* evolution and will also be helpful for the design of phytopathological control strategies.

1. Introduction

Oomycetes of the genus *Phytophthora* include devastating plant pathogens that are found in almost all ecosystems. These organisms often cause severe host infections and are responsible for considerable economic losses (Erwin and Ribeiro, 1996). Understanding the evolution of these pathogens and the associated virulence mechanisms are critical to the development of sustainable control strategies of the diseases caused by these organisms, some of which have become major epidemics (Tyler et al., 2006; Blair et al., 2008; Haas et al., 2009; Charley and David, 2017). There are 123 *Phytophthora* species (http://www.phytophtho radb.org/) which are usually soil-borne plant pathogens that cause dis ease in herbaceous and woody plants, particularly in dicotyledons. These diseases include root rot, basal stem rot, leaf spot or blight and fruit rot (Dodds and Rathjen, 2010). *Phytophthora* blights and rots are caused by oomycete pathogens having a wide host range; they infect many important crops, including red pepper, potato, tomato, alfalfa, tobacco, oaks, strawberry, and raspberry, thereby causing worldwide economic losses (Erwin and Ribeiro, 1996). *Phytophthora* species occurrence fre quencies are the highest among all plant pathogens (Gao and Zhang, 2013). Plant pathologists have therefore conducted extensive research on these pathogens and governmental agencies have developed strict con trol measures (Bae et al., 2016; EPPO, 2020).

*Phytophthora fragariae* Hickman (Hickman, 1941) and *Phytophthora rubi* (W.F. Wilcox & J.M. Duncan) (Wilcox et al., 1993) Man in't Veld (Man in 't Veld, 2007) have highly similar morphology and physiology, but infect different hosts. These two species were once defined as *Phytophthora fragariae* C.J. Hickman and *Phytophthora fragariae* var. *rubi* W.F. Wilcox & J.M. Duncan. The principal *P. fragariae* hosts are the cultivated *Fragaria × ananassa* and *Rubus ursinus* var. *longanobaccus* (Ho and Jong, 1988). For *P. rubi*, the principal hosts are the cultivated *Rubus idaeus* var. *idaeus*. Several genera within Potentilleae in Rosaceae have been artifi cially infected by both of these species (Stewart et al., 2014). *P. fragariae* and *P. rubi* were first discovered in Scotland in 1921 and 1937, respec tively (Wardlaw, 1926; Hickman, 1941). Occurrences of plant disease in the United States, Canada, Austria, France, Germany, and some other European and Mediterranean Plant Protection Organization (EPPO) re gions were previously reported. *P. fragariae* and *P. rubi* outbreaks in Australia, Cyprus, Japan, Lebanon and some Asian countries, were sub sequently reported (Pasiecznik et al., 2010), causing devastation to strawberry and raspberry productions. Both species were designated quarantine pests by many countries and regions, including EPPO regions (A2 list), the United States, and China. A recent study compared the genome structure and gene repertoires of the specific race types of *P. fragariae* and *P. rubi* and the function of several candidate avirulence effector genes was characterized (Adams et al., 2020). The genome structure, evolutionary relationships between species and pathogenesis-related factors within the *Phytophthora* genus are still

limited. Here, we sequenced the *P. fragariae* and *P. rubi* genomes and compared them to other available *Phytophthora* genomes. We investi gated the evolutionary relationships of Peronosporales and characterized gene families involved in *Phytophthora* pathogenesis, thus providing a basis for the prevention and control of these pathogens.

2. Results and discussion

2.1. Genome sequencing and characteristics

According to our K-mer analysis, the estimated actual genome sizes of *P. fragariae* 309.62 and *P. rubi* 109892 are 91.2 Mb and 103.4 Mb, respectively (Figure S1). The size of the final assembly of the *P. fragariae* genome is 75.98 Mb, with an 84.00 kb scaffold N50 and corresponding 40.50 kb contig N50 (Gao et al., 2015). The *P. rubi* genome assembly is 79.10 Mb, with an 83.99 kb scaffold N50 and corresponding 13.67 kb contig N50 (Tables S1 and S2). We annotated 25,250 and 24,528 protein-coding genes in *P. fragariae* and *P. rubi*, respectively. This iden tification was supported by transcriptomic data (Table 1; Notes S1, S2; Figures S2–5 and Tables S3, S4). The BUSCO (benchmarking universal single-copy orthologs) assessment indicated that the *P. fragariae* and *P. rubi* genome completeness was 96.1% and 95.4%, suggesting the genome assemblies are of good quality (Table S5) (Simão et al., 2015).

2.2. Genome content in Phytophthora species

We analysed *P. fragariae*, *P. rubi* and five additional available *Phy- tophthora* genomes, including *P. infestans* T30-4 (Haas et al., 2009), *P. parasitica* INRA-310 (Blackman et al., 2014), *P. sojae* V3.0 (Tyler et al., 2006), *P. nicotianae* (Liu et al., 2016a, b) and *P. ramorum* (Tyler et al., 2006). The observed size variation among these *Phytophthora* genomes can be attributed to their repeat content, length of the intergenic regions, and number of pseudogenes (Table 1 and Note S1). The repeat content proportion in *Phytophthora* species genomes varies widely, with the highest value in *P. infestans* (58.42%) and the lowest in *P. parasitica* (22.66%). It likely explains the differences in genome sizes which are following a linear correlation (Figure S6 and Table S6). Long terminal repeat retrotransposons (LTRs) are abundant transposable elements (Note S3; Figures S7, S8 and Table S6), constituting 20.45% of the *P. fragariae* genome, 13.27% of the *P. rubi* genome (Figure S8) and 45.09% of the *P. infestans* genome. The distribution in the 10-kb 5'– or 3'– regions adjacent to RxLR and CRN genes in seven *Phytophthora* species indicated that the LTR number increases proportionally with distance from RxLR and CRN (Figure S9). This finding is inconsistent with pre vious studies suggesting that RxLR and CRN are likely located near LTR-rich regions in *P. infestans* (Judelson, 2007). The impact of these repeated elements on the genome size and structure is dramatic and they may play a role in the evolution of *Phytophthora* species, including in
<table><caption>Table 1. Characteristics of seven *Phytophthora* genomes.</caption><thead><tr><th></th><th>P. fragariae</th><th>P. rubi</th><th>P. sojae</th><th>P. ramorum</th><th>P. infestans</th><th>P. parasitica</th><th>P. nicotianae</th></tr></thead><tbody><tr><td>Genome</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Estimated genome size (bp)</td><td>75,981,480</td><td>79,095,819</td><td>82,597,641</td><td>66,652,401</td><td>228,543,505</td><td>55,229,644</td><td>81,610,141</td></tr><tr><td>Coverage (fold)</td><td>214</td><td>222</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>G + C (%)</td><td>52.0</td><td>44.5</td><td>52.4</td><td>44.0</td><td>42.4</td><td>49.6</td><td>50.0</td></tr><tr><td>Repeat content length (bp)</td><td>25,741,986</td><td>18,436,422</td><td>29,710,845</td><td>14,995,776</td><td>123,280,000</td><td>11,863,539</td><td>27,724,685</td></tr><tr><td>Repeat content length (%)</td><td>33.88</td><td>23.31</td><td>35.97</td><td>22.50</td><td>53.94</td><td>21.48</td><td>33.97</td></tr><tr><td>Gene family</td><td>13906</td><td>13838</td><td>12242</td><td>9120</td><td>10276</td><td>12053</td><td>10267</td></tr><tr><td>Gene</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Protein-coding gene number</td><td>25250</td><td>24528</td><td>26489</td><td>15605</td><td>17785</td><td>23159</td><td>17348</td></tr><tr><td>Average gene length (bp)</td><td>1506.96</td><td>1416.35</td><td>1417.06</td><td>1642.1</td><td>1530.77</td><td>1729.53</td><td>1692.9</td></tr><tr><td>Average gene gap length (bp)</td><td>1339</td><td>1628</td><td>1706</td><td>1966</td><td>8440</td><td>840</td><td>1061</td></tr><tr><td>Genome coverage (%)</td><td>50.08</td><td>43.92</td><td>45.45</td><td>38.45</td><td>11.95</td><td>88.07</td><td>36.10</td></tr><tr><td>Genome density (bp/gene)</td><td>3009</td><td>3224</td><td>3118</td><td>4271</td><td>12850</td><td>2384</td><td>4704</td></tr><tr><td>mRNA</td><td>25250</td><td>24528</td><td>26489</td><td>15605</td><td>17787</td><td>28117</td><td>17348</td></tr><tr><td>tRNA</td><td>130</td><td>78</td><td>1631</td><td>140</td><td>1200</td><td>70</td><td>111</td></tr><tr><td>CDS</td><td>59680</td><td>54824</td><td>62854</td><td>40381</td><td>49146</td><td>69538</td><td>37714</td></tr><tr><td>Pseudogenes</td><td>13810</td><td>11697</td><td>25</td><td></td><td>6183</td><td>68</td><td></td></tr><tr><td>Average exon number per gene</td><td>2.36</td><td>2.24</td><td>2.37</td><td>2.59</td><td>2.78</td><td>2.47</td><td>2.17</td></tr><tr><td>Average exon length (bp)</td><td>547.55</td><td>523.06</td><td>500.62</td><td>554.16</td><td>470.72</td><td>648.28</td><td>704.30</td></tr><tr><td>Average intron number per gene</td><td>1.36</td><td>1.23</td><td>1.37</td><td>1.59</td><td>1.78</td><td>1.47</td><td>1.17</td></tr><tr><td>Average intron length (bp)</td><td>158.62</td><td>198.4</td><td>186.75</td><td>135.22</td><td>193.76</td><td>89.12</td><td>139.86</td></tr><tr><td>Total intron number</td><td>34255</td><td>30287</td><td>36365</td><td>24776</td><td>31748</td><td>41421</td><td>20366</td></tr><tr><td>Average CDS length (bp)</td><td>1290.32</td><td>1168.77</td><td>1137.48</td><td>1434</td><td>1260.66</td><td>1274.51</td><td>1531.13</td></tr></tbody></table>

facilitating them *in planta* accommodation and dampening of the host plant immune mechanisms (Raffaele et al., 2010; Gilbert and Cordaux, 2013).

We scanned the seven *Phytophthora* genomes to identify gene-dense regions (GDRs) and gene-sparse regions (GSRs) and compared their evolutionary rates (Table S7). The GSR evolutionary rate was slightly higher than GDRs in most *Phytophthora* species. First, the high GSRs evolutionary rate was consistently higher than GDRs, except in *P. fragariae*, which facilitates coevolution and provides variation during host infection (Cooke et al., 2000). Second, *P. rubi* had the highest GSRs evolutionary rate, while *P. fragariae* had the lowest evolutionary rate, with 364 and nine genes in GSRs, respectively, and only a few single-copy genes in GDRs. Third, the two main types of effectors, RxLR and CRN, were located in GSRs for *P. infestans* and *P. ramorum* (Figures S10, S11 and Table S8). This distinctive genome organization offers a unique opportunity to identify novel candidate virulence genes (Haas et al., 2009). While these effectors are always located in GDRs for *P. parasitica*. In addition, we performed a functional enrichment analysis of genes located in GSRs and found 15 enriched candidate functions for *P. ramorum*, *P. infestans*, and *P. fragariae* (Table S9). The major enriched functional categories for *P. ramorum* are those related to the cell cytoskeleton and genome structure. For *P. infestans*, enriched functions are all associated with plant defence mechanisms and the expansion of these gene categories may partly explain the destructiveness of this species. The enriched functions identified for *P. fragariae*, e.g. pectinases, may explain its wide host range and ability to infect roots.

We used 2,921 conserved, single-copy protein sequences to construct the phylogenetic tree of *Phytophthora* and close lineages (Table S10). The results show that *Plasmopara halstedii*, an economically important pathogen (Runge et al., 2011), is clustered within *Phytophthora* (Figure 1). In addition, *Hyaloperonospora arabidopsidis*, an obligate parasite that causes downy mildew and grows on only *Arabidopsis thaliana* (Coates and Beynon, 2010), is sister to *Phytophthora* spp. Estimation of the divergence time shows that *P. fragariae* and *P. rubi* diverged approximately 9.1 million years ago (Mya). Although the host of *P. fragariae* is suggested to be cultivated strawberries (*Fragaria × ananassa*), this pathogen also

infects loganberry in natural environments. The natural host of *P. rubi* is loganberry (*Rubus × loganobaccus*). The genus *Rubus* diverged from the *Fragaria* ancestor approximately 57 million years ago (MYA), long before *P. fragariae* and *P. rubi* diverged (Zhang et al., 2017). Thus, the host species change of *P. fragariae* to *Fragaria* might be a rare event, likely driven by divergence within *Rubus*.

## 2.3. *Phytophthora* genome evolution

### 2.3.1. Whole-genome duplication

WGD events have been detected in *P. infestans*, *P. sojae*, *P. ramorum*, *P. nicotianae* and *P. parasitica* genomes (Tyler et al., 2006; Haas et al., 2009; Blackman et al., 2014; Liu et al., 2016a, b). In addition, large intraspecies synteny was detected and many short homologous segments were localised on the same scaffold in *P. infestans*, *P. sojae* and *P. ramorum* (van Hooff et al., 2014). We analysed the Kolmogorov-Smirnov distributions of seven *Phytophthora* genomes. We did not observe distinct WGD signals, such as the distribution of synonymous substitutions per synonymous site (Ks), in these genomes, suggesting that recent genome duplication events did not occur in these seven species (Figure S12).

### 2.3.2. *Phytophthora*-specific gene expansion and contraction

To study the unique characteristics of *Phytophthora* and to explore the genetic features that are possibly underlying the known pathogenicity of these species, we analysed the expansion and contraction of gene families in the *Phytophthora* clade. We found 131 expanding gene families and 14 contracting families (Figure 1 and Table S11). The expanding gene families comprise two main categories: 1) genes associated with pathogenicity or pathogenic processes, and 2) genes associated with maintenance of pathogenicity or common biological processes, such as energy supply or cell structure maintenance. The enriched GO terms associated with disease mechanisms are described below. For example, we found that the gene family encoding acyl-coenzyme A oxidase (ACX) is expanded in *Phytophthora* species compared to other genera such as *Pythium*, *Plasmopara*, and *Albugo* (Schneider et al., 2005). Together with
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Phylogenetic tree correspondence with gene family expansion and contraction in oomycetes, and phylogenetic trees of seven *Phytophthora* species hosts. We constructed a phylogenetic tree (left) that included seven *Phytophthora* species (*P. fragariae*, *P. rubi*, *P. parasitica*, *P. sojae*, *P. infestans*, *P. ramorum*, and *P. nicotianae*) and other eight oomycete species, including *Hyaloperonospora arabidopsidis*, *Plasmopara halstedii*, *Pythium arrhenomanes*, *Pythium ultimum*, *Pythium iwayamai*, *Pythium irregulare*, *Albugo laibachii*, and *Albugo candida*. The phylogenetic tree of the main *Phytophthora* hosts species is shown on the right. The same colour represents correspondence between pathogen and host. Multiple colours indicate multiple correspondences. The phylogenetic tree shows the topology and divergence times for *Phytophthora* species and the corresponding hosts. Divergence times are indicated by light blue bars at the internodes. The range of these bars indicates the 95% confidence interval of the divergence time. Numbers at branches indicate the expansion and contraction of gene families.</figcaption></figure>

the CoA synthetase, ACX is acting in the β-oxidation pathway involved in the suppression of plant resistance via signalling molecules, such as jasmonic acid. Our phylogenetic analysis showed that these ACX genes could be grouped into seven clades, including the *Phytophthora*-specific clades B and C (Figure S13). No conserved specific motif was found in the protein sequences suggesting that expansion of the ACX families may facilitate *Phytophthora* infection via the increased gene copy number rather than by a specific motif. The expansion of gene family encoding flavodoxin-like proteins in *Phytophthora* may also be related to the enhanced ability of the pathogen to respond to host immunity (Li et al., 2015). Expansion of tRNA uridine 5-carboxymethylaminomethyl modification enzyme (gidA) in *Phytophthora* may play a role in cell replication, division and weaken the host immune pattern (Figure 2) (Claussen, 2005; Shippy et al., 2011). During the *Phytophthora* infection process, energy supply and channeling likely facilitates efficient invasion (Giannini et al., 1988). For example, the expansion of deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) is known to ensure an accurate DNA replication (Barabas et al., 2004).

### 2.3.3. Evolutionary origin of MADs-Box genes

A previous study showed that the type II MADS-box genes PsMADS1 from *P. sojae* and PiMADS from *P. infestans* regulate zoosporogenesis. PsMADS1 is also involved in pathogenesis (Lin et al., 2018). The present analysis of the seven *Phytophthora* genomes showed that only a single MADs-box-containing gene without a *K* domain resembles the type II MEF2-like genes in each *Phytophthora* species (Figure S14). Molecular evidence indicates that in *Phytophthora* genomes, several hundred genes were inherited from a red alga (Tyler et al., 2006), although *Phytophthora* does not contain vestigial plastids originating from phototrophs. Interestingly, four HGT (horizontal gene transfer) events occurred between *Phytophthora* and fungi, contributing to *Phytophthora* genome complexity (Tyler et al., 2006). Therefore, the origin of only one type II MADs-box gene in *Phytophthora* genomes has been identified. Phylogenetic analysis showed that oomycete, red algae, and chlorophyte type II MEF-like MADs-box proteins form a sister clade to fungi (Figure S14). The type

II MEF-like protein clade is a sister clade to the streptophyte-specific type II MIKC clade. The MRCA of streptophytes contained a protein with MADs domain similar to Type II approximately 1,000 MYA representing the ancestral MADs domain protein (Kaufmann et al., 2005). Expression analysis suggests that these genes may have a putative role in haploid reproductive cell differentiation and during the course of evolution they were recruited into a diploid generation (Thangavel, and Nayar, 2018).

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Schematic diagram depicting expanded gene families in *Phytophthora* genomes and their putative functions during host attack. Most functions are associated with attacking plant cells. The expanded gene families where it was possible to identify a putative function are represented.</figcaption></figure>
This result suggests that the MADs-box gene in *Phytophthora* might have originated from a red algae endosymbiont, but not from HGT between *Phytophthora* and fungi.

### 2.3.4. Cell wall formation

The cell wall compositions of oomycetes and fungi are chemically distinct; cellulose and chitin are the major wall components of oomycetes and fungi, respectively (Melida et al., 2013). β-1,3-glucan is another abundant polysaccharide in the oomycete wall and may play an essential role in plant pathogenesis (Raaymakers and Van den Ackerveken, 2016) as with a minor component, mannan (Hermanns and Ziegler, 1984), whose anomeric structure (α-β-) of 1,4-linkage remains unclear (Melida et al., 2013) (Figure 3).

The evolutionary trajectories of different functional classes of genes from ten Stramenopile species revealed significant expansion events in the gene classes associated with biogenesis of the cell wall, membrane and envelope in Peronosporales (*Pythium* and *Phytophthora*) but not in Saprolegniales (Seidl et al., 2012). The latter order includes mainly animal pathogens except for a few species (Gaulin et al., 2008), while plant pathogenesis of Peronosporales is thought to have evolved independently of other groups of Oomycetes (Thines and Kamoun, 2010). We conducted a phylogenetic analysis to identify cell wall synthase homologues, that is, *CesA* (cellulose) (Turner and Kumar, 2018), *CslA*, *CslD* (plant type-mannan) (Liepman and Cavalier, 2012), *Glycosyl transferase family 48* (*GT48*, β-1,3-glucan) (Stone et al., 2018), *GT32*, *GT62* and *GT71* (fungal type-mannan) (Wang et al., 2017), that diverged in the ancestral

<figure><img src="image_6.png" /></figure>

**Figure 3.** Cell wall synthesis-related genes from *Phytophthora* and their association with the pathogenic interactions with host plants. Conserved gene domains and motifs are described with the NCBI conserved domain database identification number (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). ER: endoplasmic reticulum; PM: plasma membrane; TM: transmembrane domain, *CesA*: cellulose synthase, *UDP-Glc*: UDP-glucose, *GDP-Man*: GDP-mannose, *BcsA*: conserved domain of cellulose synthase (*CesA*, NCBI conserved domain: COG1215), *Gln*: conserved β-1,3-glucan synthase domain of family 48 glycosyl transferase, *GT48* (cl15642), *CxxC1*: cysteine repeat motif similar to the zinc finger domain in oomycete *CesA3* (Blum et al., 2012), *CxxC2*: cysteine repeat motif similar to the zinc finger domain commonly found in methionine peptidase. *CxxC2* is registered as the NCBI conserved domain motif cl33650 from Viridiplantae. Major components from *Phytophthora* cell walls are cellulose and β-1,3-(1,6)-glucan (Melida et al., 2013). As a minor component, mannan was detected on the cell surface via histochemistry with lectin (Hermanns and Ziegler, 1984). The major linkage in *Phytophthora* is a 1,4 linkage (Melida et al., 2013). Cellulose is synthesised by *CesA* proteins by using UDP-glucose as a substrate (*UDP-Glc*). *Phytophthora infestans* has four *PiCesA* genes. All *PiCesAs* contain the conserved *CelA* domain, but there are various TM domains and pleckstrin homology domains (PH) or zinc finger-like motifs (*CxxC1* and 2) among the *CesA* genes. This difference could lead to different enzyme activities associated with *CesA* genes, such as the cellulose synthase-like (*Csl*): D proteins in *Arabidopsis*, which have mannan synthesis activity, although there is no report on the effects of these domains on *PiCesA* activities. Mandipropamid, a fungicide, targets cellulose synthase *PiCesA3* to inhibit cell wall biosynthesis in *P. infestans*. The main β-1,3,6-glucan backbone is synthesised by *GT48* by using *UDP-Glc* as a substrate. The *GT48* membrane protein from *Phytophthora* contains six TM domains in the N-terminal region, conserved glucan synthase-like domains (*Gns*), and eleven to twenty-two TM domains in the C-terminal regions. *Phytophthora* genomes also contain several mannosyl transferases that are homologous to *GT32* (*Och1*), *GT62* (*Amp1*), and *GT71* (*MNN1/2/5*) enzymes identified in budding yeast. These enzymes are predicted to contain an N-terminal TM domain and may localise in Golgi membranes. The catalytic domains of these enzymes (oval shapes in the figure) are located in the Golgi lumen, and the reactions require GDP-mannose supplied by nucleotide sugar transporters such as “GONST1 homologue” from the cytoplasm. *CesA* genes may be required for pathogenesis in *Phytophthora* and are also the target of carboxylic acid amides, which are oomycete fungicides. β-1,3,1,6-glucans may be a source of glucan fragments released by host β-1,3-glucanase to elicit the synthesis of the antimicrobial substance phytoalexin in host plants as a defence response. α-mannan glycoprotein from *Phytophthora megasperma* has been demonstrated to suppress the defence response in host plants stimulated by the glucan elicitor.
lineage of Peronosporales, based on a working hypothesis that Per-onosporales genes associated with plant pathogenesis should have diverged at approximately this time period (Note S4; Tables S12–15 and Figures S15–20). On the estimated phylogenetic trees, we identified a set of genes that were duplicated and already existed in the ancestral lineage of Peronosporales in *CesA*, *GT48* and *GT71* (Figures S16–20). Several duplications in these gene families occurred in the Peronosporales line-age after splitting from Saprolegniales. Some genes were duplicated more recently in different species in Peronosporales. These genes are good candidates for further functional analysis (e.g., gene inactivation) to elucidate their potential role in plant infection. Several of these genes, such as *GT71* (mannan), could be used to screen fungicides, like man-dipropamid (cellulose) (Blum et al., 2012) and poacic acid (β-1,3-glucan) (Piotrowski et al., 2015), targeting cell wall components.

### 2.3.5. Horizontal gene transfer

HGT provides a way for micro-organisms to adapt to new lifestyles, environments, and hosts (Gilbert and Cordaux, 2013). Here, we identi-fied several HGT events in the *P. fragariae* and *P. rubi* genomes via sequence similarity comparison (Figure 4 and Table S16). Transferred genes appeared to originate from plants, fungi, bacteria, mollusc and insect donors. It is inferred that most transposons in the genome may be related to horizontal gene transfer, which is able to mobile and amplify in the host genome. And horizontal transfer is an important way which would allow the element to evade a seemingly inevitable vertical extinction in its original host lineage resulting from genetic drift, natural selection or mutational inactivation. It is speculated that transposition may serve as vectors to connect exogenous genes and *Phytophthora* species, which could be compared to “transmit” to *Phytophthora* from HGT. In addition, horizontal transposon transfer in genomes has been a major

force propelling genomic variation and biological innovation. Here, candidatus HGT are inferred to encode secreted proteins, such as hy-drolase, aspartic-type endopeptidase, integrase, chalcone-flavanone isomerase, and ubiquitin-protein transferase, which may be involved in the resistance to plant defence mechanisms or maybe effector proteins that are released into plant cells. Additional HGT candidates included repeats, such as copia-type and Tc-1-like repeats. Thus, varying degrees of expansion and contraction enlarged the *Phytophthora* genome complex (Tyler et al., 2006).

### 2.3.6. Pathogenesis

*Phytophthora* species produce apoplastic effectors that are secreted into the host extracellular space and cytoplasmic effectors that are translocated into the host cytoplasm or intracellular compartments. RxLR (where x is any amino acid) is named after an N-terminal motif involved in host cell uptake (Jiang et al., 2008). CRNs are named after a “crin-kling” or necrotic phenotype that occurs when several of these proteins are overexpressed in plants (Amaro et al., 2017). We identified various candidate effectors in *Phytophthora* and found that RxLRs and CRNs are two main extracellular types. Phylogenetic analysis of the seven *Phy-tophthora* species provided information regarding species-specific fea-tures that may be related to pathogenesis (Figure 5 and Table S17).

There is a significantly higher content in RxLRs in *P. sojae*, *P. infestans*, *P. parasitica*, and *P. nicotianae*. Genome-level comparisons of RxLR genes provided evidence for diversifying selection, polymorphism, presence/absence, copy number variation, intragenic recombination, and gene conversion. CRNs are quite variable. There are far more CRNs in *P. sojae* and *P. infestans* than in other species, suggesting a potential role in the withering to death symptom observed in these two species. We screened for candidate effector gene families that were clustered into a single clade

<figure><img src="image_7.png" /><figcaption>**Figure 4.** HGT between donor and *P. fragariae* or *P. rubi*, demonstrating that most plant-, fungi-, bacteria-, mollusc-, and insect-derived genes are retained by *P. fragariae* and *P. rubi*. Using the phylogenetic analysis results in combination with alternative topology tests, we estimated the early points of transfer 23 to *P. fragariae* (red numbers) and 15 (black numbers) to *P. rubi*. The putative functions of these transferred genes are noted in the figure and listed in Table S16.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Phylogenetic tree with secreted RxLRs and CRN effector genes in seven *Phytophthora* genomes and *Plasmopara halstedii*. Phylogenetic trees were generated using maximum parsimony of amino acid sequences of *P. fragariae*, *P. rubi*, *P. parasitica*, *P. sojae*, *P. infestans*, *P. ramorum*, *P. nicotianae*, and *Plasmopara halstedii*.</figcaption></figure>

with species-level specificity during evolution (Table S18). For *P. infestans*, *CRE4* (PITG_05910T0), *CRE12* (PITG_14960T0) contributes to virulence during the early infection stage, by inhibiting plant defence responses induced by both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Yin et al., 2017). *PexRD1* (PITG_15287T0) and

*Avr-vnt11* (PITG_16294T0) enhances colonization of plant (Wang et al., 2019). *Avr3a*, *Avr2*, *PexRD8* could activate innate immunity triggered by resistance protein and is a strong suppressor of the cell-death response induced by INF1 elicitin in different level (Whisson et al., 2007; Oh et al., 2009; Gilroy et al., 2011). *SFI2* (PITG_04145T0) triggers converging
signalling pathways recruiting MAP kinase cascades and yielding a
generic antimicrobial response (Zheng et al., 2014). For *P. sojae*, series of
*Avh1b*, -96 (EGZ09809), -131 (EGZ07281), -42 (EGZ29034), -13
(EGZ20804), -31 (EGZ12148), -51 (EGZ17961), -115 (EGZ29650), -7b1
(EGZ09677), -7a (EGZ09683), -7c (EGZ09679), -16 (EGZ28985), -102
(EGZ15291), -63 (EGZ26838), -81 (EGZ30372), -66 (EGZ29639), -75
(EGZ14528), -9 (EGZ19624) are specific with the similarity of *Avr1* gene
in *P. infestans* (Jiang et al., 2008). *Avh 238* (EGZ19905) escapes host
recognition retaining plant immunity-suppressing activity to enhance
infection (Yang et al., 2018). *Avh 241* (EGZ08158) locates to the mem-
brane and triggers cell death in multiple plant species (Yu et al., 2012).
However, a large number of effectors have no known function and may
be involved in the infection mechanisms.

Simultaneously, functional enrichment analysis of RxLRs and CRNs in *Phytophthora* genomes identified several species-specific- and species-enriched candidate effectors that may be involved in the pathogenesis idiosyncrasies of each species. Enrichment was mainly reflected in several categories (Table S18 and S19): signalling pathway, receptor, ion channel, cell cytoskeleton, cellular substance, cell adhesion, motility, immunity, metabolism, cell apoptosis, and transfer. For example, with the function of signalling pathway of effectors, *P. sojae*, *P. infestans* and *P. parasitica* with specific G-protein-coupled receptor signalling but the different gene structures. In addition to different receptors, all these factors may be related to host specificity. The voltage-gated potassium channels of most *Phytophthora* species are species-specific. In *P. nicotianae*, the specific voltage-gated calcium channel may regulate the migration of zoosporogenesis, sporulation and mycelial growth (Liu et al., 2016a, b). G-protein-coupled receptor signalling in *P. sojae* and *P. infestans* are the same but are specific to gene structure, indicating that there may be different pathogenic mechanisms. Identification of the effector species specificity will aid the discovery and functional verification of new effectors.

3. Conclusions

We sequenced the *P. fragariae* and *P. rubi* genomes and combined the
data with existing genomic data for other five *Phytophthora* species.
Next, we conducted a comparative genomic analysis of genome struc-
ture, evolutionary relationships, and pathogenic characteristics of
*Phytophthora* species. Our results indicate that there were no WGD
events in these seven *Phytophthora* species. The phylogenetic relation-
ship of *Phytophthora* and other oomycete species showed that *Hyalo-
peronospora arabidopsidis* was a sister to *Phytophthora* spp. Further,
phylogenetic analysis showed that *Plasmopara halstedii* was a member
of *Phytophthora*. Comparison of gene repertoires suggested that signal
recognition, membrane barrier disruption, host defence weakening,
and auxiliary effector molecule activity are associated with the Phy-
tophthora infection and pathogenicity. Molecular evidence indicates
that in *Phytophthora* genomes, several hundred genes are putatively
inherited from red algae, although *Phytophthora* species do not have
vestigial plastids originating from phototrophs. We characterized
several expanding *Phytophthora* gene families associated with cell wall
biogenesis. Further functional analysis is required to elucidate the
function of these genes in pathogenic interactions. It may be postulated
that HGTs in *P. fragariae* and *P. rubi* from plants, fungi, bacteria,
molluscs, and insects might impact genes those are involved in plant
defense mechanisms. Some of these genes encode effector proteins
that are released in plant cells to manipulate the defence reactions.
Analysis of RxLRs and CRN sequences in seven *Phytophthora* genomes
identified genes that may be related to the pathogenesis specificity of
each species. Species-specific distribution of genes involved in signal-
ling pathways, receptors, ion channels, cell cytoskeleton, cellular sub-
stances, cell adhesion, motility, immunity, metabolism, cell apoptosis,
and transfer were also identified. Our results provide a genetic basis for
understanding the evolution of these pathogens and the associated

virulence mechanisms, which are key to the development of sustainable
disease control strategies.

4. Methods

4.1. Materials availability

The *P. fragariae* and *P. rubi* strains were imported from Westerdijk
Fungal Biodiversity Institute (strain numbers 309.62 and 109892,
respectively). *P. fragariae* 309.62 was isolated from *Fragaria* fruit by C.J.
Hickman in Scotland, and *P. rubi* 109892 was isolated by C. Brasier from
raspberry roots in Scotland.

4.2. Strain culture

We induced the production of motile zoospores in non-sterile pond
water for up to 5 h at 13-14 °C and pH values of 6.0-6.8. Single zoospores
were picked by inoculating needles under a microscope, followed by
germination in V8 plates at 18 °C. After 48 h, the mycelia began to form
and were collected in 5-6 days. The mycelia (~100 mg) were ground to a
fine powder under liquid nitrogen. Genomic DNA was extracted using the
Qiagen DNeasy Plant Mini Kit (Qiagen, Germany) according to the
manufacturer's protocol.

4.3. Sequencing and assembly

We sequenced and assembled the genome of *P. fragariae* 309.62 and
*P. rubi* 109892 using a strategy that combined paired-end and mate-
paired libraries. Two paired-end libraries, each with a targeted insert
size of 180 bp and 500 bp for *P. fragariae* 309.62 and of 180 bp and 300
bp for *P. rubi* 109892, were constructed using the TruSeq Nano DNA LT
Library Prep Kit (Illumina, USA), and two mate-paired libraries (3 kb and
5 kb) were constructed for each genome using the Nextera Mate Pair
Sample Prep Kit (Illumina, FC-132-1001, USA) according to the manu-
facturer's protocol. Both genomes were sequenced on the Illumina HiSeq
2500 platform. De novo assembly was performed using Allpaths-LG
(Butler et al., 2008). The assembled genome scaffolds were aligned to
the most closely related publicly available genomes using MUMmer
(Edgar, 2004a, b), which included the three previously published Phy-
tophthora complete genomes, namely, *P. infestans* T30-4 (Haas et al.,
2009, Wang et al., 2017), *P. parasitica* INRA-310 (Blackman et al., 2014)
and *P. sojae* V3.0 (Tyler et al., 2006).

Scaffolds were broken at points where non-contiguous regions of the
reference genome were juxtaposed and then re-ordered so that the
scaffolds were syntenic with the reference genome. All scaffolds from a
given strain were concatenated into a single pseudogenome, separated by
the sequence NNNNCACACACTTAATTAATTAAGTGTGTGNNNNN,
which contained stop codons in all six reading frames. Scaffolds that did
not match the reference genomes were concatenated in random order at
the end of the genome. The pseudogenomes were annotated using the
RAST automated annotation server. The genome sequences of the newly
sequenced strains were deposited into GenBank.

4.4. Genome annotation

Protein-coding genes were identified by the MAKER pipeline, which
integrated *ab initio* gene prediction and protein and expressed sequence
tag (EST) homology (Cantarel et al., 2008), and subjected to manual
correction using an in-house script (HiCESAP). Gene structures were
initially automatically annotated using a combination of gene models
predicted by using the gene search programs Augustus (Stanke et al.,
2004; Stanke and Morgenstern, 2005), GeneMark-ES (Lomsadze et al.,
2005) and SNAP (Korf, 2004). Protein family and orthologue clustering
were performed using TribeMCL (Dongen, 2000) and OrthoMCL (Fischer
et al., 2011), respectively, using matching protein pairs (BLASTP,
E<=1e-10). Protein domains and product names were assigned based on
sequence homology to known proteins or Pfam domains with scores greater than the trusted cutoff scores (Bateman et al., 2004). Predicted gene models were submitted to GO (Harris et al., 2004), KEGG, Swiss-prot, TrEmbl, Interpro for functional annotation. Gene functional categories were computed by searching annotated proteins against the NCBI COG and KOG databases using BLASTP ($E \le 1e-10$) and transitively assigning functional categories based on the single best match. RNA genes were predicted using both tRNA-scanSE and Rfam. Gene families corresponding to CRN proteins, NPP1-like proteins, elicitin-like proteins, small secreted cysteine-rich proteins (SCRs), and transporters were targeted for manual review. Evidence for gene identification and editing of exon boundaries was derived from protein and EST alignments based on related proteins of known function. In the case of NPP1-like proteins, elicitins, and transporters, matches to corresponding Pfam profiles were used for identification of genomic loci, and GeneWise (run with the pseudo option) was used to aid pseudogene identification. Several bioinformatics strategies have been previously employed for identification of candidate RxLR effectors within genome sequences, relying on matches to a hidden Markov model (HMM) profile or to a sequence pattern (regular expression or 'regex').

### 4.5. Repetitive elements

Repeat sequences were identified using two homologous searches (RepeatMasker version 3.3.0 and the Repbase database version 20120418) and *ab initio* annotations (RepeatModeler, Tandem Repeats Finder, and LTR-finder). Additionally, the high numbers of pseudogenes in both *P. fragariae* 309.62 and *P. rubi* 109892 may be a consequence of the expansion of repeat families. RIP (repeat-induced point mutation) was a self-protective mechanism developed by lower eukaryotes. Although it was generally considered to be fungus-specific, we cannot rule out that this mechanism may also occur in oomycetes. The Pearson test was used for the correlation of repeat length and repeat ration components (Venkat et al., 2017).

### 4.6. Comparative genomic analysis

We used OrthoMCL (version 2.0.8) to identify single-copy genes in related species, and then, MUSCLE (version 3.8.425) was used to align the sequences of the associated proteins (Edgar, 2004a, b). PAL2NAL (version 14.0) was used to convert the protein alignment to a codon alignment (Suyama et al., 2006). Gblock (version 0.91b) was used to remove the alignment results that were deemed unreliable (Castresana, 2000). Finally, the codeml program in PAML (version 4.7) was used to calculate $d_N$ (non-synonymous) and $d_S$ (synonymous) substitutions (Yang, 2007). The one-ratio model (M0) was used to estimate the overall selection pressure in the species examined, and the free-ratio model (M1) was used to estimate the $d_N/d_S$ ($\omega$) ratio in a certain branch. We then used KEGG and GoSlim for functional enrichment analysis of the obtained orthologue groups that satisfy certain criteria.

For gene family analysis, we first downloaded all the protein sequences for the studied reference genomes and used this data set as the query for all-vs-all BLASTP, with the cutoff value set at $1e^{-10}$. OrthoMCL was used to filter the query result, with an alignment length of at least 70% of the query sequences, and MCL was used to cluster the gene families, with I (inflation) set at 1.5. CAFÉ (computational analysis of gene family evolution, version 3.1) was used to calculate the expansion and contraction of these gene families (Hahn et al., 2005). A phylogenetic tree was also constructed based on the single-copy genes in the whole genome, using *P. halstedii* as the outgroup. After the Gblock step, ProtTest (version 3.2) (Darriba et al., 2011) was used to select the best amino acid substitution model (JTT, BIC = 17073927.92), and then, PhyML was used to build the tree. MCMCTree software in PAML (version 4.7) was used to estimate the divergence time between close species (Guindon et al., 2010). The following constraints were used for time calibrations: (i) the *P. infestans* and *P. parasitica* divergence time (2–22 (MYA)); (ii) the

*P. infestans* and *Pythium ultimum* divergence time (70–80 MYA); (iii) the *P. infestans* and *A. laibachii* divergence time with a lower boundary of 135 MYA. All the data above are collected by Timetree Database (http://www.timetree.org/). Mauve (version 2.3.1) was used to align two closely related genomes for synteny analysis (Darling et al., 2010). The functional domains in each genome were identified using Pfam (http://pfam.xfam.org/) and InterProScan (Jones et al., 2014).

### 4.7. Whole-genome duplication

According to the method described by Martens and Van de Peer (2010), the number of synonymous substitutions per synonymous site (Ks) in whole *Phytophthora* genomes was analysed. Protein sequences were subjected to intra-species BLASTP alignment, and pairs of genes that were optimally aligned (RBH) were selected as homologous gene pairs for the species. MUSCLE software was used to perform multi-sequence alignment of homologous proteins and to convert the results to a nucleic acid alignment. Then, codeml in the Paml package was used to calculate the Ks value, and Ks values greater than 5 were filtered out to generate a distribution curve. The Ks distributions generated from the *Phytophthora* genomes all lacked a distinct peak, and it was concluded that there was no evidence of WGD or large-scale duplication in these seven genomes.

### 4.8. GSRs and GDRs

According to the method of Haas et al. (2009), in the study of *P. infestans*, with a cutoff L value of 1.5 kb, the core orthologous genes of seven *Phytophthora* genomes were classified as GDRs and GSRs. The gene families of GSRs and GDRs in *P. fragariae* and *P. rubi* were statistically significant (Table S7). GO functional enrichment of GSRs in *P. ramorum*, *P. infestans* and *P. fragariae* was performed (Table S9).

### 4.9. Analysis of MADS-box genes

MADS-box genes were identified by searching the InterProScan (Zdobnov and Apweiler, 2001) results of all the predicted *Phytophthora* proteins. The predicted genes were manually inspected, and the predicted genes were short or the MADs domains were only partially included. MADs-box domains comprising 60 amino acids, identified by SMART (Letunic et al., 2015) for all the MADs-box genes, were then aligned using ClustalW. An unrooted maximum likelihood phylogenetic tree was constructed in MEGA7 with default parameters (Kumar et al., 2016). Bootstrap analysis was performed using 1,000 iterations.

### 4.10. Cell wall formation

We acquired the predicted protein sequences by performing BLAST or keyword search against the following eight databases in addition to the sequences of the seven *Phytophthora* species (*P. fragariae*, *P. infestans*, *P. parasitica*, *P. ramorum*, *P. rubi*, *P. nicotianae*, *P. sojae*):

1) https://bioinformatics.psb.ugent.be/plaza/versions/pico-plaza/

2) https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins

3) http://www.cazy.org/GlycosylTransferases.html

4) https://phytozome.jgi.doe.gov/pz/portal.html

5) http://bioinformatics.psb.ugent.be/blast/moderated/?project=orcae_Chbra

6) http://www.plantmorphogenesis.bio.titech.ac.jp/%7Ealgae_genome_project/klebsormidium/klebsormidium_blast.html

7) https://www.UniProt.org/

8) https://www.genome.jp/

Data sets for NCBI protein BLAST search were selected according to taxon name—Chlorophyta (taxid: 3041), Heterokonta (taxid: 33634), Oomycota (taxid: 4762), Peronosporales (taxid: 4776), Rhodophyta
(taxid: 2763), and Saprolegniales (taxid: 4763)—in addition to each species name. Accession numbers of the collected sequences were provided (Table S13).

The acquired protein sequences were analysed by NCBI conserved domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to extract the conserved sequences of the *CesA*, *GT48*, *GT62* and *GT71* families, which were used for phylogenetic analysis. The *GT32* sequences were analysed with the “phobius” program (http://phobius.sbc.su.se/) (Kall et al., 2007) for prediction of the transmembrane (TM) domains. We used the predicted catalytic regions of *GT32* without the N-terminal TM domain for the phylogenetic analysis. The edited sequences were provided in Table S14, which also included the “CxxCT” and “CxxC2” motif sequences described in Figure S15. The edited sequences were analysed again with the NCBI conserved domain search, and the results are shown in Table S15.

The conserved domains of the GT protein sequences were edited as described above and used for phylogenetic tree estimation. Each set of homologous sequences was aligned using mafft (v7, G-INS-I algorithm) (Katoh and Standley, 2013). The resulting alignment, excluding gapped sites, was used to infer a maximum likelihood phylogenetic tree using PhyML v3.1 with the gamma model of heterogeneity and the estimated alpha parameter assuming the WAG amino acid substitution model. The robustness of the tree topology was assessed using 1,000 bootstrap replicates. The resulting tree was visualized with MEGA version 6 (Tamura et al., 2013).

## 4.11. HGT

According to the clustering results for fungi, the unique gene families of *P. fragariae* and *P. rubi*, as well as the non-clustered genes, were found to together constitute the unique genes of this species. These unique genes were not clustered with other oomycetes but may be acquired via horizontal transfer and are defined as HGT candidate genes. Subsequently, the sequences of these candidate genes were aligned to the non-redundant (nr) plant, fungal, bacterial, mollusc and insect libraries to identify a candidate gene capable of acting as donor genes that were horizontally transferred. The candidate genes generated in the second part were aligned with the NCBI nr library for optimal alignment of the donor genes, which were considered to be relatively reliable horizontal transferred genes. According to the method of Tyler et al., (2006) in the study of *P. sojae* and *P. ramorum*, we statistically analysed the horizontally transferred genes of *P. fragariae* and *P. rubi* from plants, fungi, bacteria, molluscs and insects (Table S16).

## 4.12. Effectors

The RxLR HMM file "pf16810.hmm" was downloaded from Pfam. A protein with the same conserved region in the species as the protein set directly via hmm search was the RxLR protein of the species. For CRN prediction, multiple protein sequences of *Phytophthora* CRNs were downloaded from the NCBI protein database. Then, multiple sequence alignment of the *Phytophthora* CRN protein sequences was performed using MUSCLE 3.8 software, and an HMM of the CRN proteins was constructed using the hmmbuild program in the hmmer 3.1b2 software package.

## Declarations

### Author contribution statement

Rui-Fang Gao, Zhong-Jian Liu, Gui-Ming Zhang: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Jie-Yu Wang: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Ke-Wei Liu, Kouki Yoshida, Yu-Yun Hsiao, Yi-Xiang Shi, Kun-Chan Tsai, You-Yi Chen, Nobutaka Mitsuda, Chieh-Kai Liang, Zhi-Wen Wang, Di-Yang Zhang, Laiqiang Huang, Xiang Zhao, Wen-Ying Zhong, Ying-Hui Cheng, Ming-He Li, Wei-Hong Sun, Xia Yu, Wenqi Hu, Zhuang Zhou, Xiao-Fan Zhou: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Ying Wang, Francis Martin: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Zi-De Jiang, Chuan-Ming Yeh, Kazutaka Katoh, Wen-Chieh Tsai: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

### Funding statement

This work was supported by National Key R&D Program of China (Grant No. 2019YFD1000400) and the Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization Construction Funds of Fujian, China (Grant Nos. 115/118990050 and 115/KJG18016A) for Z.-J.L., and the National Key Technology Research and Development Programme of China (Grant No. 2012BAK11B06) and National Key R&D Programme of China (Grant No. 2016YFF0203204) for G.-M. Z. The cell wall analyses were partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED (Grant No. JP19am0101108) for K.K.

### Data availability statement

Data included in article/supp. material/referenced in article.

### Competing interest statement

The authors declare no conflict of interest.

### Additional information

Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2020.e03459.

## References

Adams, T.M., Armitage, An.D., Sobczyk, M.K., Bates, H.J., Tabima, J.F., Kronmiller, B.A., Tyler, B.M., Grünwald, N.J., Dunwell, J.M., Nellist, C.F., et al., 2020. Genomic investigation of the strawberry pathogen *Phytophthora fragariae* indicates pathogenicity is determined by transcriptional variation in three key races. *Front. Microbiol.* 11, 1–17.

Amaro, T.M.M.M., Thiliez, G.J.A., Motion, G.B., Huitema, F., 2017. A perspective on CRN proteins in the genomics age: evolution, classification, delivery and function revisited. *Front. Plant Sci.* 8, 99.

Bae, S.J., Mohanta, T.K., Chung, J.Y., Ryu, M., 2016. Trichoderma metabolites as biological control agents against *Phytophthora* pathogens. *Biol. Contr.* 92, 128–138.

Bateman, A., Coin, L., Durbin, R., Finn, R., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E., et al., 2004. The Pfam protein families database. *Nucleic Acids Res.* 32, D138–D141.

Barabas, O., Pongracz, V., Kovari, J., Wilmanns, M., Vertessy, B.G., 2004. Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase. *J. Biol. Chem.* 279, 42907–42915.

Blackman, L.M., Cullerne, D.P., Hardham, A.R., 2014. Bioinformatic characterisation of genes encoding cell wall degrading enzymes in the *Phytophthora parasitica* genome. *BMC Genom.* 15, 785.

Blair, J.E., Coffey, M.D., Park, S.Y., Geiser, D.M., Kang, S., 2008. A multi-locus phylogeny for *Phytophthora* utilizing markers derived from complete genome sequences. *Fungal Genet. Biol.* 45, 266–277.

Blum, M., Gamper, H.A., Waldner, M., Sierotzki, H., Gisi, U., 2012. The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides. *Fungal Biol.* 116, 529–542.

Butler, J., MacCallum, I., Kleber, M., Shlyakhter, I.A., Belmonte, M.K., Lander, E.S., Nusbaum, C., Jaffe, D.B., 2008. ALLPATHS: de novo assembly of whole-genome shotgun microreads. *Genome Res.* 18, 810–820.
Cantarel, B.L., Korf, I., Robb, S.M., Parra, G., Ross, E., Moore, B., Holt, C., Sánchez, A.A., Yandell, M., 2008. MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res. 18, 188–196.

Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552.

Charley, G.P., David, A., 2017. Phylogenomic reconstruction of the oomycete phylogeny derived from 37 genomes. Msp2e 2, e00095, 17.

Claussen, W., 2005. Proline as a measure of stress in tomato plants. Plant Sci. 168, 241–248.

Cooke, D.E.L., Drenth, A., Duncan, J.M., Wagels, G., Brasier, C.M., 2000. A molecular phylogeny of *Phytophthora* and related oomycetes. Fungal Genet. Biol. 30, 17–32.

Coates, M.E., Beynon, J.L., 2010. *Haloferonospora arabidopsidis* as a pathogen model. Annu. Rev. Phytopathol. 48, 329–345.

Darling, A.E., Mau, B., Perna, N.T., 2010. Progressivemaue: multiple genome alignment with gene gain, loss and rearrangement. PloS One 5, e11147.

Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2011. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164–1165.

Dodds, P.N., Rathjen, J.P., 2010. Plant immunity: towards an integrated view of plant-pathogen interactions. Nat. Rev. Genet. 11, 539–548.

Dongen, S.M.V., 2000. Graph clustering by Flow Simulation. Ph.D. thesis. University of Utrecht, Utrecht.

Edgar, R.C., 2004a. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinf. 5, 113.

Edgar, R.C., 2004b. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797.

**EPPO, 2020. EPPO A2 list, 9. https://www.eppo.int/ACTIVITIES/plant_quarantine/A2_list.**

Erwin, D.C., Ribeiro, O.K., 1996. *Phytophthora* Disease Worldwide. Department of Plant Pathology, University of California, Riverside, USA.

Fischer, S., Brunk, B.P., Chen, F., Gao, X., Stoeckert, C.J., 2011. Using OrthoMCL to assign proteins to OrthoMCL-DB groups or to cluster proteomes into new ortholog groups. Curr. Protoc. Bioinform. Chapter 6, Unit 6.12.11–19.

Gao, R.F., Zhang, G.M., 2013. Potential of DNA barcoding for detecting quarantine fungi. Phytopathology 103, 1103–1107.

Gao, R.F., Cheng, Y.H., Wang, Y., Guo, L.Y., Zhang, G.M., 2015. Genome Sequence of *Phytophthora fragariae* var. *fragariae*, a quarantine plant-pathogenic fungus. Genome Announc. 3, 25–30.

Gaulin, E., Madoui, M.A., Bottin, A., Jacquet, C., Mathé, C., Couloux, A., Wincker, P., Dumas, B., 2008. Transcriptome of *Aphanomyces euteiches*: new oomycete putative pathogenicity factors and metabolic pathways. PloS One 4, e1723.

Giannini, J.L., Holt, J.S., Briskin, D.P., 1988. Isolation of sealed plasma membrane vesicles from *Phytophthora megasperma* f. sp. glycinea. I. Characterization of proton pumping and ATPase activity. Arch. Biochem. Biophys. 265, 337–345.

Gilbert, C., Cordaux, R., 2013. Horizontal transfer and evolution of prokaryote transposable elements in eukaryotes. Genome Biol. Evol. 5, 822–832.

Gilroy, E.M., Breen, S., Whisson, S.C., Squires, J., Hein, I., Kaczmarek, M., Turnbull, D., Boevink, P.C., Lokossou, A., Cano, L.M., et al., 2011. Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in *Phytophthora infestans* determine virulence on R2 plants. New Phytol. 191, 763–776.

Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321.

Haas, B.J., Sophien, K., Zody, M.C., Jiang, R.H.Y., Handsaker, R.E., Cano, L.M., Grabherr, M., Kodira, C.D., Raffaele, S., Torto-Alalibu, T., et al., 2009. Genome sequence and analysis of the Irish potato pathogen *Phytophthora infestans*. Nature 461, 393–398.

Hahn, M.W., Bie, T.J., Stajich, J.E., Nguyen, C., Cristianini, N., 2005. Estimating the tempo and mode of gene family evolution from comparative genomic data. Genome Res. 15, 1153–1160.

Harris, M.A., Clark, J., Ireland, A., Lomax, J., Ashburner, M., Foulger, R., Eilbeck, K., Lewis, S., Marshall, B., Mungall, C., et al., 2004. The gene ontology (GO) database and informatics resource. Nucleic Acids Res. 32, D258–D261.

Hermanns, R., Ziegler, E., 1984. Localization of α-mannan in the hyphal wall of *Phytophthora megasperma* f. sp. glycinea and its possible relevance to the host-pathogen interaction of the fungus with soybeans (Glycine max). J. Phytopathol. 109, 363–366.

Hickman, C.J., 1941. The red core root disease of the Strawberry caused by *Phytophthora fragariae*. n.sp. J. Pomol. Hortic. Sci. 18, 89–118.

Ho, H., Jong, S., 1988. *Phytophthora fragariae*. Mycotaxon 31, 305–322.

Jiang, R.H.Y., Tripathy, S., Govers, F., Tyler, B.M., 2008. RXLR effector reservoir in two *Phytophthora* species is dominated by a single rapidly evolving superfamily with more than 700 members. Proc. Natl. Acad. Sci. U.S.A. 105, 4874–4879.

Jones, P., Binns, D., Chang, H.Y., Fraser, M., Li, W.Z., McAnulla, C., McWilliam, H., Maslen, J., Mitchell, A., Nuka, G., et al., 2014. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240.

Judelson, H.S., 2007. Genomics of the plant pathogenic oomycete *Phytophthora*: insights into biology and evolution. Adv. Genet. 57, 97–141.

Kall, I., Krogh, A., Sonnhammer, E.L., 2007. Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server. Nucleic Acids Res. 35, W429–W432.

Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780.

Kaufmann, K., Melzer, R., Theissen, G., 2005. MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants. *Gene* 347, 183–198.

Korf, I., 2004. Gene finding in novel genomes. BMC Bioinf. 5, 59.

Piotrski, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.

Letunic, I., Doerks, T., Bork, P., 2015. SMART: recent updates, new developments and status in 2015. Nucleic Acids Res. 43, D257–D260.

Li, L., Naseem, S., Sharma, S., Konopka, J.B., 2015. Flavodoxin-like proteins protect candida albicans from oxidative stress and promote virulence. PLoS Pathog. 11, e1005147.

Liepmann, A.H., Cavalier, D., 2012. The cellulose synthase-like A and cellulose synthase-like C families: recent advances and future perspectives. Front. Plant Sci. 3, 109.

Lin, L., Ye, W.W., Wu, J.W., Xuan, M.R., Li, Y.F., Gao, J., Wang, L., Wang, Y., Dong, S.M., Wang, Y.C., 2018. The MADS-box transcription factor PsMAD1 is involved in zoosporogenesis and pathogenesis of *Phytophthora sojae*. Front. Microbiol. 9, 2259.

Liu, H., Ma, X., Yu, H.Q., Fang, D.H., Li, Y.P., Wang, X., Wang, W., Dong, Y., Xiao, B.G., 2016a. Genomes and virulence difference between two physiological races of *Phytophthora nicotianae*. GigaScience 5, 3.

Liu, P.Q., Gong, J., Ding, X.L., Jiang, Y., Chen, G.L., Li, B.J., Weng, Q.Y., Chen, Q.H., 2016b. The L-type Ca²⁺ channel blocker nifedipine inhibits mycelial growth, sporulation, and virulence of *Phytophthora capsici*. Front. Microbiol. 7, 1236.

Lomsadze, A., Ter-Hovhannisyan, V., Chernoff, Y.O., Borodovsky, M., 2005. Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res. 33, 6494–6506.

Man in 't Veld, W.A., 2007. Gene flow analysis demonstrates that *Phytophthora fragariae* var. rubi constitutes a distinct species, *Phytophthora rubi* comb. nov. Mycologia 99, 222–226.

Martens, C., Van de Peer, Y., 2010. The hidden duplication past of the plant pathogen *Phytophthora* and its consequences for infection. BMC Genom. 11, 353.

Melida, H., Sandoval-Sierra, J.V., Dieguez-Uribeondo, J., Bulone, V., 2013. Analyses of extracellular carbohydrates in oomycetes unveil the existence of three different cell wall types. Eukaryot. Cell 12, 194–203.

Oh, S.K., Young, C., Lee, M.Y., Oliva, R., Bozkurt, T.O., Cano, L.M., Win, J., Bos, J.I.B., Liu, H.Y., van Damme, M., et al., 2009. In planta expression screens of *Phytophthora infestans* RXLR effectors reveal diverse phenotypes, including activation of the *Solanum bulbocastanum* disease resistance protein Rpi-blb2. Plant Cell 21, 2928–2947.

Pasiecznik, N.M., Smith, I.M., Watson, G.W., Brunt, A.A., Charles, L.M.F., 2010. CABI/EPPO distribution maps of plant pests and plant diseases and their important role in plant quarantine. EPPO Bull. 35, 1–7.

Piotrowski, J.S., Okada, H., Lu, F., Li, S.C., Hinchman, L., Ranjan, A., Smith, D.L., Higbee, A.J., Ulbrich, A., Coon, J.J., et al., 2015. Plant-derived antifungal agent poaic acid targets β-1,3-glucan. Proc. Natl. Acad. Sci. U.S.A. 112, e1490–e1497.

Raaymakers, T.M., Van den Ackerveken, G., 2016. Extracellular recognition of oomycetes during biotrophic infection of plants. Front. Plant Sci. 7, 906.

Raffaele, S., Farrer, R.A., Cano, L.M., Studholme, D.J., MacLean, D., Thines, M., Jiang, R.H.Y., Zody, M.C., Kunjeti, S.G., Donofrio, N.M., et al., 2010. Genome evolution following host jumps in the Irish potato famine pathogen lineage. Science 330, 1540–1543.

Runge, F., Telle, S., Ploch, S., Savory, E., Day, B., Sharma, R., Thines, M., 2011. The inclusion of downy mildews in a multi-locus-dataset and its reanalysis reveals a high degree of paraphyly in *Phytophthora*. *IMA Fungus* 2, 163–171.

Schneider, K., Kienow, L., Schmelzer, E., Colby, T., Bartsch, M., Miersch, O., Wasternack, C., Kombrink, E., Stuible, H.P., 2005. A new type of peroxisomal acyl-coenzyme A synthetase from *Arabidopsis thaliana* has the catalytic capacity to activate biosynthetic precursors of jasmonic acid. J. Biol. Chem. 280, 13962–13972.

Seidl, M.F., Ackerveken, G.V.D., Govers, F., Snel, B., 2012. Reconstruction of oomycete genome evolution identifies differences in evolutionary trajectories leading to present-day large gene families. Genome Biol. Evol. 4, 199–211.

Shippy, D.C., Eakley, N.M., Bochsler, P.N., Chopra, A.K., Fadl, A.M., 2011. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene. Microb. Pathog. 50, 303–313.

Schimá, F.A., Waterhous, R.M., Ioannidis, P., Kriventseva, E.V., Zdobnov, E.M., 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 19, 3210–3212.

Stanke, M., Morgenstern, B., 2005. AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res. 33, W465–W467.

Stanke, M., Steinkamp, R., Waack, S., Morgenstern, B., 2004. AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res. 32, W309–W312.

Stewart, J.E., Kroese, D., Tabima, J.F., Larsen, M.M., Grünwald, J.N., 2014. Pathogenicity, fungicide resistance, and genetic variability of *Phytophthora rubi* isolates from raspberry (*Rubus idaeus*) in the western United States. Plant Dis. 98, 1702–1708.

Stone, B.A., Jacobs, A.K., Hrmova, M., Burton, R.A., Fincher, G.B., 2018. Biosynthesis of plant cell wall and related polysaccharides by enzymes of the GT2 and GT48 families. Annu. Plant Rev. Online 14, 109–165.

Suyama, M., Torrents, D., Bork, P., 2006. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 34, W609–W612.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

Thangavel, G., Nayar, S., 2018. A survey of mikc type mads-box genes in non-seed plants: algae, bryophytes, lycophytes and ferns. Front. Plant Sci. 18, 510.
Thines, M., Kamoun, S., 2010. Oomycete-plant coevolution: recent advances and future prospects. Curr. Opin. Plant Biol. 13, 427-433.

Turner, S., Kumar, M., 2018. Cellulose synthase complex organization and cellulose microfibril structure. Philos. Trans. R. Soc. A 376, 20170048.

Tyler, B.M., Tripathy, S., Zhang, X.M., Dehal, P., Jiang, R.H.Y., Aerts, A., Arredondo, F.D., Baxter, L., Bensasson, D., Beynon, J.L., et al., 2006. *Phytophthora* genome sequences uncover evolutionary origins and mechanisms of pathogenesis. *Science* 313, 1261-1266.

van Hooff, J.J., Snel, B., Seidl, M.F., 2014. Small homologous blocks in *Phytophthora* genomes do not point to an ancient whole-genome duplication. *Genome Biol. Evol.* 6, 1079-1085.

Venkat, T., Alexander, S., Faheema, K., Vlad, D., Roger, V., Magne, F., Christer, W., Niclas, B., 2017. Rapid increase in genome size as a consequence of transposable element hyperactivity in wood-white (Leptidea) butterflies. *Gen. Biol. Evol.* 9, 2491-2505.

Wang, P., Wang, H., Gai, J.T, Tian, X.L., Zhang, X.X, Lv, Y.Z., Jian, Y., 2017. Evolution of protein N-glycosylation process in Golgi apparatus which shapes diversity of protein N-glycan structures in plants, animals and fungi. Sci. Rep. 7, 40301.

Wang, S.M., McLellan, H., Bukharova, T., He, Q., Murphy, F., Shi, J.Y., Sun, S.H., van Weymers, P., Ren, Y.J., Thilliez, G., et al., 2019. *Phytophthora infestans* RXLR effectors act in concert at diverse subcellular locations to enhance host colonization. J. Exp. Bot. 70, 343-356.

Wardlaw, C., 1926. Lanarkshire Strawberry Disease. A Report for the Use of Growers. Botany Department, University of Glasgow, Glasgow.

Whisson, S.C., Boevink, P.C., Moleleki, L., Avrova, A.O., Morales, J.G., Gilroy, E.M., Armstrong, M.R., Grouffaud, S., van West, P., Chapman, S., et al., 2007.

A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450, 115-118.

Wilcox, W.F., Scott, P.H., Hamm, P.B., Kennedy, D.M., Duncan, J.M., Brasier, C.M., Hansen, E.M., 1993. Identity of a *Phytophthora* species attacking raspberry in Europe and North America. *Mycol. Res.* 97, 817-831.

Yang, B., Wang, Y.Y., Guo, B.D., Jing, M.F., Zhou, H., Li, Y.F., Wang, H.N., Huang, J., Wang, Y., Ye, W.W., et al., 2018. The *Phytophthora sojae* RXLR effector Avh238 destabilizes soybean Type2 GmACSs to suppress ethylene biosynthesis and promote infection. *New Phytol.* 222, 425-437.

Yang, Z., 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586-1591.

Yin, J.L., Gu, B., Huang, G.Y., Tian, Y., Quan, J.L., Lindqvist-Kreuze, H., Shan, W.X., 2017. Conserved RXLR effector genes of *Phytophthora infestans* expressed at the early stage of potato infection are suppressive to host defense. *Front. Plant Sci.* 8, 2155.

Yu, X.L., Tang, J.L., Wang, Q.Q., Ye, W.W., Tao, K., Duan, S.Y., Lu, C.C., Yang, X.Y., Dong, S.M., Zheng, X.B., et al., 2012. The RxLR effector Avh241 from *Phytophthora sojae* requires plasma membrane localization to induce plant cell death. *New Phytol.* 196, 247-260.

Zdobnov, E.M., Apweiler, R., 2001. InterProScan-an integration platform for the signature-recognition methods in InterPro. *Bioinformatics* 17, 847-848.

Zhang, S.D., Jin, J.J., Chen, S.Y., Chase, M.W., Soltis, D.E., Li, H.T., Yang, J.B., Li, D.Z., Yi, T.S., 2017. Diversification of Rosaceae since the late cretaceous based on plastid phylogenomics. *New Phytol.* 214, 1355-1367.

Zheng, X.Z., Fraiture, M., Liu, X.Y., Chen, Y., Brunner, F., 2014. Functionally redundant RXLR effectors from *Phytophthora infestans* act at different steps to suppress early flg22-triggered immunity. *PLoS Pathog.* 10, e1004057.
**Update**

Heliyon

Volume 7, Issue 6, June 2021, Page

DOI: https://doi.org/10.1016/j.heliyon.2021.e07332
Corrigendum

Corrigendum to “Comparative analysis of Phytophthora genomes reveals oomycete pathogenesis in crops” [Heliyon 7 (2) (February 2021) e06317]

<figure><img src="image_9.png" /></figure>

Rui-Fang Gao<sup>a,b,1</sup>, Jie-Yu Wang<sup>c,d,1</sup>, Ke-Wei Liu<sup>e,f,g,1</sup>, Kouki Yoshida<sup>h,1</sup>, Yu-Yun Hsiao<sup>i,1</sup>, Yi-Xiang Shi<sup>j</sup>, Kun-Chan Tsai<sup>k</sup>, You-Yi Chen<sup>l</sup>, Nobutaka Mitsuda<sup>m</sup>, Chieh-Kai Liang<sup>l</sup>, Zhi-Wen Wang<sup>n</sup>, Ying Wang<sup>a,b</sup>, Diyang Zhang<sup>c</sup>, Laiqiang Huang<sup>e,f</sup>, Xiang Zhao<sup>n</sup>, Wen-Ying Zhong<sup>n</sup>, Ying-Hui Cheng<sup>o</sup>, Zi-De Jiang<sup>p</sup>, Ming-He Li<sup>c</sup>, Wei-Hong Sun<sup>c</sup>, Xia Yu<sup>c</sup>, Wenqi Hu<sup>c</sup>, Zhuang Zhou<sup>c,q</sup>, Xiao-Fan Zhou<sup>p</sup>, Chuan-Ming Yeh<sup>m,r,s,**</sup>, Kazutaka Katoh<sup>t,***</sup>, Wen-Chieh Tsai<sup>i,l,u,****</sup>, Zhong-Jian Liu<sup>c,f,q,v,*</sup>, Francis Martin<sup>w,*****</sup>, Gui-Ming Zhang<sup>a,b,******</sup>

DOI of original article: https://doi.org/10.1016/j.heliyon.2021.e06317.

E-mail addresses: chuanmingy@nchu.edu.tw (C.-M. Yeh), katoh@ifrec.osaka-u.ac.jp (K. Katoh), tsaiwc@mail.ncku.edu.tw (W.-C. Tsai), zjliu@fafu.edu.cn (Z.-J. Liu), francis.martin@inrae.fr (F. Martin), zgm2001cn@163.com (G.-M. Zhang).

<footnote><sup>a</sup> Animal & Plant Inspection and Quarantine Technology Center of Shenzhen Customs District P.R. China, Shenzhen 518045, China</footnote>

<footnote><sup>b</sup> Shenzhen Key Laboratory for Research & Development on Detection Technology of Alien Pests, Shenzhen Academy of Inspection and Quarantine, Shenzhen 518045, China</footnote>

<footnote><sup>c</sup> Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization at College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou 350002, China</footnote>

<footnote><sup>d</sup> Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China</footnote>

<footnote><sup>e</sup> School of Life Sciences, Tsinghua University, Beijing 100084, China</footnote>

<footnote><sup>f</sup> Center for Biotechnology and Biomedicine, Shenzhen Key Laboratory of Gene and Antibody Therapy State Key Laboratory of Health Sciences and Technology (prep), Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China</footnote>

<footnote><sup>g</sup> Center for Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute (TBSI), Shenzhen 518055, China</footnote>

<footnote><sup>h</sup> Technology Center, Taisei Corporation, Nase-cho 344-1, Totsuka-ku, Yokohama, Kanagawa 245-0051, Japan</footnote>

<footnote><sup>i</sup> Orchid Research and Development Center, National Cheng Kung University, Tainan 701, Taiwan</footnote>

<footnote><sup>j</sup> Shanghai Major Bio-pharm Technology Co., Ltd., Shanghai 201203, China</footnote>

<footnote><sup>k</sup> Reber Genetics Co., Ltd., Taipei 10685, Taiwan</footnote>

<footnote><sup>l</sup> Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan</footnote>

<footnote><sup>m</sup> Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan</footnote>

<footnote><sup>n</sup> PubBio-Tech Services Corporation, Wuhan 430070, China</footnote>

<footnote><sup>o</sup> Fairylake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen 518004, China</footnote>

<footnote><sup>p</sup> College of Agriculture, South China Agricultural University, Guangzhou 510640, China</footnote>

<footnote><sup>q</sup> Zhejiang Institute of Subtropical Crops, Zhejiang Academy of Agricultural Sciences, Wenzhou 325005, China</footnote>

<footnote><sup>r</sup> Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan</footnote>

<footnote><sup>s</sup> Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan</footnote>

<footnote><sup>t</sup> Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan</footnote>

<footnote><sup>u</sup> Institute of Tropical Plant Sciences and Microbiology, National Cheng Kung University, Tainan 701, Taiwan</footnote>

<footnote><sup>v</sup> Institute of Vegetable and Flowers, Shandong Academy of Agricultural Sciences, 250100, Jinan, China</footnote>

<footnote><sup>w</sup> Institut National de la Recherche Agronomique, UMR Interactions Arbres/Microorganismes, Centre INRA Grand Est-Nancy, Université de Lorraine, 54280 Champenoux, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>*** Corresponding author.</footnote>

<footnote>**** Corresponding author.</footnote>

<footnote>***** Corresponding author.</footnote>

<footnote>****** Corresponding author.</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
In the original published version of this article, some of the language was misleading and allowed for misinterpretation of the article. In order to fix this issue, the following revisions have been made:

In the Abstract, the sentence "The horizontally-transferred Phytophthora genes are abundant transposons that "transmit" exogenous gene to Phytophthora species thus bring about the gene recombination possibility" has been revised to "The horizontally-transferred Phytophthora genes are from abundant transposon activities that "transmit" exogenous genes to Phytophthora species and thus bring about the gene recombination possibility."

In 2.3.5 Horizontal gene transfer, the paragraph "Most pervasive HGT genes are transposons, which is able to mobile and amplify in the host genome, which make them more prone to horizontal transfer. And horizontal transfer is an important way which would allow the element to evade a seemingly inevitable vertical extinction in its original host lineage resulting from genetic drift, natural selection or mutational inactivation. It was result that transposition "transmitting" foreign genes to Phytophthora species from HGT" has been revised to "It is inferred that

most transposons in the genome may be related to horizontal gene transfer, which is able to mobile and amplify in the host genome. And horizontal transfer is an important way which would allow the element to evade a seemingly inevitable vertical extinction in its original host lineage resulting from genetic drift, natural selection or mutational inactivation. It is speculated that transposition may serve as vectors to connect exogenous genes and Phytophthora species, which could be compared to "transmit" to Phytophthora from HGT."

In 3. Conclusions, "Horizontally transferred genes in P. fragariae and P. rubi from plants, fungi, bacteria, molluscs, and insects are often transposons that impact genes involved in plant defence resistance mechanisms. Some" has been revised to "It may be postulated that HGTs in P. fragariae and P. rubi from plants, fungi, bacteria, molluscs, and insects might impact genes those are involved in plant defense mechanisms."

The authors apologize for the errors. Both the HTML and PDF versions of the article have been updated to correct the errors.