<table><caption>TABLE 3 | Continued</caption><thead><tr><th colspan="2">Prot ID</th><th>Gravy</th><th>Length</th><th>%G</th><th>%CC</th><th>Score</th><th>Blast</th><th>Stress induction</th><th>Proposed name</th></tr></thead><tbody><tr><td>Abra07543</td><td>AB04946</td><td>-1.41</td><td>59</td><td>8.5</td><td>64</td><td>285</td><td></td><td></td><td></td></tr><tr><td>Abra07677</td><td>AB05060</td><td>-1.35</td><td>281</td><td>11.4</td><td>63</td><td>1535</td><td></td><td></td><td></td></tr><tr><td>Abra07755</td><td>AB05132</td><td>-1.18</td><td>425</td><td>9.2</td><td>61</td><td>231</td><td></td><td></td><td></td></tr><tr><td>Abra07777</td><td>AB05148</td><td>-1.08</td><td>132</td><td>10.6</td><td>70</td><td>437</td><td></td><td>+</td><td>AbSih15</td></tr><tr><td>Abra07844</td><td>AB05207/</td><td>-1.09</td><td>177</td><td>10.2</td><td>78</td><td>529</td><td></td><td></td><td></td></tr><tr><td>Abra08036</td><td>AB05365 AB05364</td><td>-1.00</td><td>1314</td><td>10.3</td><td>68</td><td>7</td><td>DHN3</td><td>+</td><td>AbDhn3</td></tr><tr><td>Abra08116</td><td>AB05433</td><td>-1.24</td><td>383</td><td>18.5</td><td>62</td><td>3040</td><td></td><td>+</td><td>AbSih16</td></tr><tr><td>Abra08144</td><td>AB05453</td><td>-1.10</td><td>131</td><td>16</td><td>74</td><td>1964</td><td></td><td>+</td><td>AbSih17</td></tr><tr><td>Abra08312</td><td>AB01538</td><td>-1.25</td><td>178</td><td>12.4</td><td>70</td><td>2200</td><td></td><td></td><td></td></tr><tr><td>Abra08733</td><td>AB06615</td><td>-1.15</td><td>265</td><td>9.1</td><td>63</td><td>216</td><td></td><td></td><td></td></tr><tr><td>Abra09230</td><td>AB08588</td><td>-1.22</td><td>148</td><td>8.1</td><td>60</td><td>22</td><td></td><td></td><td></td></tr><tr><td>Abra09944</td><td>AB07063</td><td>-1.55</td><td>93</td><td>8.6</td><td>53</td><td>99</td><td></td><td></td><td></td></tr><tr><td>Abra09988</td><td>AB07108</td><td>-1.29</td><td>108</td><td>9.3</td><td>58</td><td>298</td><td></td><td></td><td></td></tr><tr><td>Abra10303</td><td>AB09288</td><td>-1.17</td><td>233</td><td>9.4</td><td>54</td><td>97</td><td></td><td></td><td></td></tr><tr><td>Abra10788</td><td>AB07911</td><td>-1.34</td><td>139</td><td>12.9</td><td>63</td><td>2186</td><td></td><td></td><td></td></tr><tr><td>Abra10847</td><td>AB07968</td><td>-1.21</td><td>76</td><td>10.5</td><td>68</td><td>953</td><td></td><td></td><td></td></tr><tr><td>Abra10981</td><td>AB08081</td><td>-1.13</td><td>206</td><td>8.7</td><td>51</td><td>9</td><td></td><td></td><td></td></tr><tr><td>Abra11043</td><td>AB08134</td><td>-1.18</td><td>127</td><td>12.6</td><td>62</td><td>1008</td><td></td><td></td><td></td></tr><tr><td>Abra11399</td><td>AB06102</td><td>-1.40</td><td>238</td><td>20.6</td><td>75</td><td>12640</td><td></td><td></td><td></td></tr><tr><td>Abra11512</td><td>AB06021</td><td>-1.51</td><td>189</td><td>11.1</td><td>65</td><td>2379</td><td></td><td></td><td></td></tr><tr><td>Abra11736</td><td>AB05830</td><td>-1.31</td><td>188</td><td>13.3</td><td>81</td><td>5016</td><td></td><td></td><td></td></tr><tr><td>Abra11761</td><td>AB05809</td><td>-1.73</td><td>94</td><td>9.6</td><td>62</td><td>1393</td><td></td><td></td><td></td></tr><tr><td>Abra12091</td><td>AB05542</td><td>-1.01</td><td>384</td><td>8.3</td><td>76</td><td>9</td><td></td><td></td><td></td></tr><tr><td>Abra12172</td><td>AB05473</td><td>-1.88</td><td>224</td><td>12.1</td><td>81</td><td>11218</td><td></td><td></td><td></td></tr><tr><td>Abra12188</td><td>AB10143</td><td>-1.18</td><td>326</td><td>10.4</td><td>80</td><td>1281</td><td></td><td></td><td></td></tr><tr><td>Abra12207</td><td>AB10131</td><td>-1.03</td><td>101</td><td>14.9</td><td>58</td><td>186</td><td>HSP12</td><td>+</td><td>AbSih18</td></tr><tr><td>Abra12251</td><td>AB10094</td><td>-1.23</td><td>242</td><td>13.6</td><td>62</td><td>1522</td><td></td><td></td><td></td></tr><tr><td>Abra09652</td><td></td><td>-1.27</td><td>62</td><td>11.3</td><td>72</td><td>1944</td><td></td><td></td><td></td></tr><tr><td>Abra11726</td><td></td><td>-1.09</td><td>109</td><td>8.3</td><td>51</td><td>3</td><td></td><td></td><td></td></tr><tr><td>Abra02927</td><td></td><td>-1.03</td><td>75</td><td>13.3</td><td>57</td><td>114</td><td>con 6</td><td></td><td></td></tr><tr><td>Abra04037</td><td></td><td>-1.06</td><td>103</td><td>8.7</td><td>52</td><td>8</td><td></td><td></td><td></td></tr><tr><td>Abra04374</td><td></td><td>-1.57</td><td>62</td><td>9.7</td><td>88</td><td>3709</td><td></td><td></td><td></td></tr><tr><td>Abra04664</td><td></td><td>-1.14</td><td>180</td><td>14.4</td><td>62</td><td>1092</td><td></td><td></td><td></td></tr><tr><td>Abra05405</td><td></td><td>-1.47</td><td>104</td><td>10.6</td><td>64</td><td>1722</td><td></td><td></td><td></td></tr><tr><td>Abra06548</td><td></td><td>-1.45</td><td>164</td><td>10.4</td><td>59</td><td>971</td><td></td><td></td><td></td></tr><tr><td>Abra12282</td><td></td><td>-1.46</td><td>159</td><td>12.6</td><td>60</td><td>2115</td><td></td><td></td><td></td></tr><tr><td>Abra00223</td><td></td><td>-1.47</td><td>70</td><td>8.6</td><td>54</td><td>113</td><td></td><td></td><td></td></tr><tr><td>Abra00730</td><td></td><td>-1.28</td><td>63</td><td>11.1</td><td>63</td><td>1126</td><td></td><td></td><td></td></tr><tr><td>Abra01125</td><td></td><td>-1.01</td><td>44</td><td>9.1</td><td>79</td><td>44</td><td></td><td></td><td></td></tr><tr><td>Abra02379</td><td></td><td>-1.01</td><td>73</td><td>20.5</td><td>53</td><td>41</td><td></td><td></td><td></td></tr><tr><td>Abra02426</td><td></td><td>-1.67</td><td>52</td><td>13.5</td><td>57</td><td>2562</td><td></td><td></td><td></td></tr><tr><td>Abra04371</td><td></td><td>-1.83</td><td>68</td><td>20.6</td><td>85</td><td>36512</td><td></td><td></td><td></td></tr><tr><td>Abra04903</td><td></td><td>-1.44</td><td>69</td><td>8.7</td><td>53</td><td>93</td><td></td><td></td><td></td></tr><tr><td>Abra05665</td><td></td><td>-1.05</td><td>60</td><td>8.3</td><td>65</td><td>22</td><td></td><td></td><td></td></tr><tr><td>Abra06247</td><td></td><td>-1.06</td><td>51</td><td>9.8</td><td>62</td><td>131</td><td></td><td></td><td></td></tr><tr><td>Abra07265</td><td></td><td>-1.56</td><td>52</td><td>9.6</td><td>71</td><td>1880</td><td></td><td></td><td></td></tr><tr><td>Abra07528</td><td></td><td>-1.00</td><td>70</td><td>15.7</td><td>72</td><td>48</td><td></td><td></td><td></td></tr><tr><td>Abra07587</td><td></td><td>-1.11</td><td>51</td><td>9.8</td><td>64</td><td>267</td><td></td><td></td><td></td></tr><tr><td>Abra08211</td><td></td><td>-1.32</td><td>54</td><td>9.3</td><td>51</td><td>41</td><td></td><td></td><td></td></tr><tr><td>Abra10089</td><td></td><td>-1.39</td><td>62</td><td>11.3</td><td>62</td><td>1552</td><td></td><td></td><td></td></tr><tr><td>Abra10521</td><td></td><td>-1.52</td><td>52</td><td>9.6</td><td>71</td><td>1732</td><td></td><td></td><td></td></tr></tbody></table>

(Continued)
<table><caption>**TABLE 3** | Continued</caption><thead><tr><th colspan="2">Prot ID</th><th rowspan="2">Gravy</th><th rowspan="2">Length</th><th rowspan="2">%G</th><th rowspan="2">%CC</th><th rowspan="2">Score</th><th rowspan="2">Blast</th><th rowspan="2">Stress induction</th><th rowspan="2">Proposed name</th></tr></thead><tbody><tr><td>Ab43 genome</td><td>ref genome</td><td>-1.52</td><td>52</td><td>9.6</td><td>71</td><td>1732</td><td></td><td></td><td></td></tr><tr><td>Abra10564</td><td></td><td>-1.40</td><td>58</td><td>8.6</td><td>72</td><td>526</td><td></td><td></td><td></td></tr><tr><td>Abra11431</td><td></td><td>-1.10</td><td>52</td><td>9.6</td><td>53</td><td>47</td><td></td><td></td><td></td></tr><tr><td>Abra12286</td><td></td><td>-2.04</td><td>66</td><td>10.6</td><td>69</td><td>5127</td><td></td><td></td><td></td></tr><tr><td>Abra07568</td><td></td><td>-1.01</td><td>56</td><td>12.5</td><td>85</td><td>225</td><td></td><td></td><td></td></tr></tbody></table>

<figure><img src="image_74.png" /></figure>

350 mM NaCl, 1.2 M sorbitol) was found compared to the wild-type (data not shown).

Consistently, a similar higher susceptibility to H₂O₂ of the *Sa. cerevisiae* *hsp12* null mutant was observed (Figure 6). To confirm that *AbSih3* correspond to true functional homolog of the yeast *hsp12* gene, complementation of *hsp12* mutant null mutant was carried out with relevant recombinant pYES plasmids. As shown in Figure 6, *AbSih3* successfully complemented the *Sa. cerevisiae* *hsp12* phenotypes, even if the growth of *Δhsp12* cells expressing *AbSih3* was not restored to a level comparable to that of the WT strain containing the empty vector pYES2 on media supplemented with 1 mM H₂O₂.

Then, the virulence of the *A. brassicicola* wild-type, *Δabsih3* and *Δabsih15* strains were compared on *B. oleracea* and *A. thaliana* host plants during host vegetative tissue infection or during the seed transmission process. *Brassica oleracea* leaves were inoculated with drops of conidia suspension (10⁵, 10⁴, or 10³ conidia/mL). As shown in Figure 7, the wild-type and KO strains were all able to produce typical symptoms and their aggressiveness was not impaired. Regardless of the inoculated strain, first necrotic symptoms appeared on leaves at 3 days post-inoculation (dpi). Necrotic areas enlarged and surrounded

themselves with chlorotic halos at 6 dpi. During late stages of infection, the fungus produced conidia on the surface of necrosis.

Using the model pathosystem previously described by Pochon et al. (2012) for investigating seed transmission in *Arabidopsis* plants, the ability of the hydrophilin mutants to transmit to seeds was compared with that of the wild-type. As a positive control, we also analyzed the behavior of the osmosensitive fungal mutants *Δabnik1* and *Δabhog1*, which were previously described as being strongly altered in their seed transmission capacity (Pochon et al., 2012). Silique inoculation with the wild-type and hydrophilin mutant strains resulted in the development of mycelium and typical lesions covered with conidia on siliques within a few days after inoculation. Seeds were then individually harvested and plated on PDA medium for a microbiological examination. As expected, a strong decrease in seed transmission rate was observed for *Δabnik1* and *Δabhog1* compared to the wild-type parental strain (Figure 8). Interestingly, the seed transmission capacity of the *Δabsch9* mutant was not altered although this strain was found to be sensitive to sorbitol and desiccation. While the deletion of *AbSih3* had a slight significant impact on seed transmission, a stronger decrease was observed for the *Δabsih15* mutant, for which the seed
<figure><img src="image_75.png" /></figure>

**FIGURE 5** | Growth inhibition rates of the wild-type strain (WT) and hydrophilin deficient mutants in the presence of H₂O₂ (10 mM) and menadione (20 mM). The results are expressed as the percentage of inhibition in treated samples compared to the control without additive. Conidia were used to inoculate microplate wells containing standard PDB medium that was supplemented with the appropriate test substance. Growth was automatically recorded for 30 h at 25°C using a nephelometric reader (see section "Materials and Methods"). Each genotype was analyzed in triplicate and the experiments were repeated at least three times per growth condition. Error bars indicate standard deviations. Asterisks indicate a significant difference between the mutant and the parental isolate (Dunn control test, \*p ≤ 0.01).

transmission rate was reduced by a factor two compared to the
wild-type strain.

DISCUSSION

The ability of *A. brassicicola* to cope with water-stress
constitutes a key factor for successful seed colonization and thus
accomplishment of its entire infectious cycle (Iacomi-Vasilescu
et al., 2008; Pochon et al., 2012, 2013). Highly hydrophilic and
disordered glycin-rich proteins containing repeated DPR-motifs
called dehydrins were identified in *A. brassicicola* and might
correspond to effectors of the response to water stress (Pochon
et al., 2013). Based on their physicochemical features, dehydrins
are considered as members of a larger protein family called
hydrophilins initially described in *Sa. cerevisiae* (Garay-Arroyo
et al., 2000). In an attempt to identify additional effectors of the
response of *A. brassicicola* to water stress, the proteome deduced
from the recently published genome of *A. brassicicola* strain
Abra43 (Belmas et al., 2018) was first screened for proteins with
characteristic features of hydrophilins. Candidate hydrophilins
accounted for 0.9% of the whole predicted proteome. In

comparison, hydrophilins represented only 0.2% of the yeast
proteome (Garay-Arroyo et al., 2000) although the selection
criteria were less stringent, i.e., proteins with lower glycine
content were included. This difference might reflect a family
expansion associated with environmental adaptation and/or
fungal pathogenesis as it was proposed for other sets of proteins,
e.g., secreted proteins, enzymes for secondary metabolites
production (Soanes et al., 2008; Ohm et al., 2012). However, this
assumption remains to be demonstrated and would require a
more extensive search for hydrophilin repertoires in fungi with
various lifestyles.

The fact that two of the three previously described dehydrins
and additional homologs of fungal hydrophilins were included in
the repertoire of candidate hydrophilins validated our screening
process. Garay-Arroyo et al. (2000) proposed that hydrophilins
that were not induced by water-stress conditions should not be
considered as true hydrophilins. Thus, we analyzed changes in
transcript levels in *A. brassicicola* germlings exposed to two kinds
of water-stress: hyperosmosis (exposure to 1.2 M sorbitol) and
desiccation (exposure to silica gel beads). Using gene expression
array, which was designed from a previous genome sequence
with a lower quality, we defined the expression patterns of 78
genes (over 107) encoding these putative hydrophilins. Twenty
candidate hydrophilin genes were up-regulated either by the
sorbitol treatment or by the desiccation stress, and, among them,
ten were induced by both treatments. So, according to the
hydrophilin definition given by Garay-Arroyo et al. (2000), we
identified at least 20 hydrophilins in *A. brassicicola*. However, we
can reasonably assume that their number is higher since we did
not have access to the gene expression of 29 other genes encoding
putative hydrophilins.

In this study, strains deficient for AbSih3 exhibited a slightly
lower susceptibility to oxidative stress compared to the wild-
type strain. No significant difference in susceptibility to other
tested stresses (exposure to PEG, NaCl and sorbitol) was
found for Δ*absih3* and Δ*absih15*. Given the large number of
hydrophilins that were co-expressed in this organism, functional
redundancy may occur between some of these proteins. This
process would explain the scarcity of significant phenotypes that
we obtained from single deficient mutants since the presence
of other hydrophilin genes would compensates for the loss. For
instance, AbSih3 displays high sequence similarity to AbSih18
(AB10131), and both may be considered as homologs of the
*Sa. cerevisiae* HSP12 hydrophilin, even if AbSih3 only partially
compensated the HSP12 loss in yeast. The existence of such
a functional redundancy was also previously observed between
two dehydrins AbDhn1 and AbDhn2 in *A. brassicicola*. The
double mutant ΔΔ*abdhn1-abdhn2* was severely compromised in
its pathogenicity while the respective single mutants were not
affected (Pochon et al., 2013).

Despite this probable functional compensation, we showed
here that the hydrophilins Absih3 and Absih15 could be
considered as pathogenic factors since the corresponding null
strains were affected in its seed transmission ability (more
strongly for the Δ*absih15* strain). This result confirmed our initial
assumption that an efficient fungal adaptive response to severe
hydric stress conditions or to oxidative stress strongly influenced
<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

**FIGURE 6** | Complementation of yeast Δhsp12 mutants with AbSih3 coding sequences. (A) Growth curves of *Saccharomyces cerevisiae* (parental WT strain and Δhsp12 mutants) cells transformed with empty vector (pYES2CT) or recombinant vectors (pYAbsih3) exposed to 1 mM H₂O₂ or to water. (B) Comparisons of growth inhibition rates obtained for each yeast strain (Wilcoxon test, \*p < 0.001).

<figure><img src="image_78.png" /></figure>

**FIGURE 7** | Representative symptoms at 5 dpi obtained by inoculation of wildtype, Δ*absch9*, Δ*absih3*, and Δ*absih15* on cabbage leaves. Leaves were inoculated with 5 µL drops of conidia suspension (10⁵, 10⁴ or 10³ conidia/mL in water). As shown on the right side, mutants were inoculated on the right part of the central vein and compared on the same leaf with the parental strain (inoculated on the left part of the central vein). Bars = 2 cm.

the seed transmission ability. This is consistent with the fact that, during seed colonization, fungi are exposed to a gradual decrease in the water potential occurring in maturing reproductive organs and that they have to cope with this particular stress to be efficiently transmitted to seeds and complete their infection cycle. In the same vein, we showed in this study or in previous works that the class III HK AbNik1 and the MAP kinase AbHog1, two components of the high osmolarity pathway, were required for an effective seed colonization by *A. brassicicola* (Pochon et al., 2012). The Δ*absch9* mutant was also found to be sensitive to sorbitol and desiccation but, unexpectedly, its seed transmission capacity was not altered. The fact that this mutant strain did not exhibit a high susceptibility to oxidants suggested that the fungal ability to overcome oxidative stress would be an additional or the main key mechanism for an efficient seed infection process. This hypothesis is consistent with the fact that oxidative damages produced by free radical species are considered to be one of the major causes of desiccation injuries (Dupont et al., 2014).

A final point raised from these results is that several *A. brassicicola* mutants (Δ*abnik1*, Δ*absih3* and Δ*absih15*) were affected in their transmission to *A. thaliana* seeds, although

their aggressiveness on host vegetative tissues remained intact. This fact confirms the specificity of some molecular mechanisms controlling transgenerational transmission of fungal pathogens (Pochon et al., 2012) and validates the strategy to gain further insight into these mechanisms for the development of specific seed disease control strategies.

In addition to the induction of genes encoding hydrophilins, we identified other metabolic clues related to the activation of an adaptive response to water stress in *A. brassicicola*. First, the cellular content in amino acids was strongly disturbed following the application of both stresses. The amino acid levels were indeed significantly reduced in treated cells compared to controls. In line with this result, several enriched GO categories emerging from transcriptomic data were linked to amino acid metabolism and catabolism. Moreover, amino acid synthesis was also found to be strongly down-regulated in response to the osmotic stress treatment in another *Dothideomycete* fungus, the wheat pathogen *Stagonospora nodorum* (Lowe et al., 2008). As reported in plants (Hildebrandt et al., 2015), the degradation of amino acids may lead to the production of an additional energetic resource, that could be useful to cope with adverse conditions, by
<figure><img src="image_79.png" /></figure>

**FIGURE 8** | Transmission capacity of *Alternaria brassicicola* wild-type (WT) and null strains to *Arabidopsis thaliana* seeds (Ler ecotype). The seed transmission capacity were measured as described by Pochon et al. (2012). The five youngest siliques of at least five plants were inoculated with each fungal genotype and the experiment was repeated twice. Contaminated siliques were harvested 10 dpi. After dissection, seeds were incubated separately on PDA medium for 2 days. A seed was considered contaminated when incubation resulted in typical *Alternaria brassicicola* colony development. For each inoculated fungal genotype, the seed infection probability was evaluated from at least 1000 seeds. Values represent infection probabilities with 95% confidence interval.

providing intermediates of the tricarboxylic acid cycle and finally contributing to ATP production. Moreover, the oxidation of some amino acids feeds electrons into the mitochondrial electron transport chain. Most reaction steps of the catabolic pathways occur either in the mitochondria or cytosol. Consistent with this hypothesis, several enriched GO terms referred to mitochondria organization, carboxylic acid metabolism and to oxidation-reduction process. Regarding the response to desiccation, as a GO enrichment of functions related to the genesis and transport of ribosomes and, more generally, to translation was observed, we could assume that a strong induction of the translation process would trigger a depletion of the cellular amino acid pool. So, the mechanisms leading to the decrease of the amino acid could depend on the type of applied stress.

An intriguing result from our data was that no significant difference in the amino acid concentrations was observed for the 1 h-desiccation treatment compared to the respective control. A more precise inspection showed that, for this condition, both control and treated samples showed a similar low amino acid content (Supplementary Figure S2). In this assay, germlings grown on cellophane were taken off from the PDA plate and directly transferred to sealed boxes (without additional agar medium) with or without silica gel beads. In the control box (without silica gel), we suspect that germlings were also exposed

to a strong constraint in the early time of exposure, as a period of 4 h was required to gradually reach a RH value of about 90%. Therefore, although this experimental device triggered a specific regulation at the transcriptome level (Figures 1, 2), this does not appear to be sufficient to reveal significant differences in amino acid accumulation between control and treated samples at this analysis time.

In the sorbitol-exposed samples, a strong increase in global sugar contents was observed compared to sorbitol control. Interestingly, the accumulation of non-reducing disaccharides, such as trehalose, did not seem to be favored in *A. brassicicola* germlings under the sorbitol stress conditions. On the contrary, the contents of three polyols, glycerol, arabitol and dulcitol, were mainly impacted by this stress, suggesting that they function as intracellular osmolytes to counteract the effects of decreases in the cell volume and the loss of turgor pressure (Sánchez-Fresneda et al., 2013; Dupont et al., 2014). This result was consistent with the increased sensitivity of the Δ*abhog1* mutant to osmotic stress since the MAP kinase Hog1 is known to be required for endogenous glycerol production in several fungal species (Brewster and Gustin, 2014; Hohmann, 2015). However, its involvement in the synthesis of other protective compounds, such as arabitol or dulcitol, is still unclear (Kayingo and Wong, 2005; Sánchez-Fresneda et al., 2013). Arabitol has been described as part of a set of polyols which is developed to counteract environmental challenges in several yeasts and filamentous fungi (Pascual et al., 2003; Sánchez-Fresneda et al., 2013). For instance, arabitol, and to a lesser extent glycerol, were found to accumulate in response to the osmotic stress treatment in the wheat pathogen *St. nodorum* (Lowe et al., 2008) or in the rice pathogen *Magnaporthe grisea* (Dixon et al., 1999). Conversely, the role of dulcitol in stress responses remains highly confidential and has been reported on rare occasions, for instance in the marine fungus *Cirrenalia pygmea* (Ravishankar et al., 2006).

In addition to its involvement in the synthesis of compatible solutes, we showed here that the HOG pathway, mainly regulated by the MAP kinase AbHog1 and the upstream HK AbNik1, also acted as an expression regulator of some hydrophilin encoding genes. The expression profiles of hydrophilin genes in the Δ*abhog1*, Δ*abnik1*, and Δ*absch9* mutants were indeed compared with those in the wild-type parental strain after exposure to sorbitol for 0.5 h. Of the 17 proteins that were induced under these conditions, 11 and 5 were found to be under the control of AbHog1 and AbNik1, respectively. This result, associated with the weakness of Δ*abnik1* and Δ*abhog1* to survive on reproductive organs, highlighted that the HOG pathway is a major target for controlling both the synthesis of osmolytes and hydrophilins, and thus limiting the transmission of fungal pathogens to seeds. Unexpectedly, AbDhn1 and AbDhn3 were not part of this set of HOG-regulated proteins although Pochon et al. (2013) previously reported that these two proteins (in addition to AbDhn2) were targets for the AbHog1 signaling cascade when the fungus was exposed to a saline (NaCl) stress. This suggests a possible differential regulation of hydrophilins depending on the applied constraint.
Following a 0.5 h-sorbitol exposure, six other hydrophilins were also found to be AbSch9-dependent. As a major downstream effector of the target of rapamycin (TOR) complex 1, the Sch9 protein kinase was proposed to play multiple roles in stress resistance and nutrient sensing in *Sa. cerevisiae* (Pascual-Ahuir and Proft, 2007; Huber et al., 2009). However, the functions of *Sch9* orthologs in filamentous fungi have been poorly characterized to date. In *Fusarium graminearum*, Gu et al. (2015) showed that FgSch9 served as a mediator of the TOR and HOG pathways and played important roles in regulating vegetative differentiation, secondary metabolism and stress responses. We showed here that both hydrophilins AbSih3 and AbSih15 were necessary factors for an optimal *A. brassicicola* transmission to seeds but their expression was not found to be regulated by AbSch9 (regulation by AbHog1 and AbHog1-AbNik1, respectively). This result, associated with the fact that the seed transmission capacity of the *Δabsch9* mutant was not affected, suggested that this kinase (unlike AbHog1 and AbNik1), although involved in resistance to sorbitol and desiccation, is not a major player in transgenerational transmission of fungal pathogens.

# MATERIALS AND METHODS

## Strains and Culture Conditions

The *A. brassicicola* wild-type strain *Abra43* used in this study was previously described (Sellam et al., 2007). For routine culture, *A. brassicicola* was grown and maintained on potato dextrose agar (PDA). The genome of *Abra43* strain was recently published and is available following this link https://bbric-pipelines.toulouse.inra.fr/myGenomeBrowser? (Carrere and Gouzy, 2017).

For transcriptomic analyses, exposure to sorbitol (osmotic stress) or to low relative humidity (desiccation stress) were achieved as follows. Conidia were first incubated for 24 h on cellophane disks overlaid on solid PDA medium. Germlings and the underlying cellophanes were then taken off from the agar plate and transferred either to a second PDA medium supplemented or not with 1.2 M sorbitol for 0.5 or 2 h.

The desiccation stress was achieved by applying a drastic decrease in relative humidity (RH) to the germlings (obtained as described below) by exposure to silica gel beads in sealed boxes for 0.5 or 4 h. Immediately after deposition of germlings (and the underlying cellophanes) in boxes, a strong RH increase was observed. In the control box (without silica gel), a gradual increase up to a RH value of about 90% in 4 h was observed. With silica gel beads, the opening of the boxes resulted in a rapid RH increase up to 20% followed by a decrease to a RH value of 1% in less than 0.5 h.

To evaluate the level of stress for each treatment applied to fungal cells and considering that the reference strain is naturally tolerant to water stress (Iacomi-Vasilescu et al., 2008), we additionally analyzed the effect of treatments on three mutant strains (derived from the wild-type *Abra43* strain) for which we hypothesized a higher sensitivity to water stress. Two mutant strains (*Δabnik1* and *Δabhog1*) were described

in previous studies (Dongo et al., 2009; Joubert et al., 2011) and were deficient for genes encoding key regulators of the HOG pathway, the HK AbNik1 and the MAP kinase AbHog1, respectively. The third mutant was obtained in this study and was deficient for the kinase AbSch9, which is also known to be involved in the fungal response to osmotic constraints (Pascual-Ahuir and Proft, 2007). Under non-stress conditions, no significant differences in mycelium growth were detected in any of the tested mutants as compared to the wild-type (data not shown). To assess the impact of low RH exposure on *A. brassicicola*, viability tests were conducted on young hyphae of wild-type and mutant strains. About 200 conidia were first incubated for 24 h on cellophane disks overlaid on solid PDA medium. Then, germinated conidia were transferred for 4 days to sealed boxes containing or not silica gel beads. The cellophane membranes were then removed from the boxes and deposited again on PDA medium. The survival rate was calculated by counting the number of mycelial colonies (derived from treated or untreated germlings) after 3 days of incubation at 24°C. As previously reported (de Lima Alves et al., 2015; Wang et al., 2017), exposure to these two treatments induces water stress in fungal cells. Exposure of wild-type and mutant strains to 1.2 M sorbitol were performed following the procedure described below. The radial growth was calculated after 6 days of incubation.

To study hyphal growth in liquid media, conidial suspensions ($10^5$ conidia/mL, final concentration) were inoculated onto microplate wells containing the considered substance ($10$ mM $H_2O_2$ or $20$ mM menadione) at the desired concentrations in potato dextrose broth (PDB, Difco) in a total volume of $300~\mu L$. Fungal growth was monitored automatically over a 30 h period with a laser-based microplate nephelometer (NEPHELOstar, BMG Labtech) as described by Joubert et al. (2010). For each condition, the area under the growth curve representative of the lag phase was used to calculate the percentages of growth inhibition under stress conditions compared with standard growth conditions (PDB medium without additive), as described by Gaucher et al. (2013). At least three replicates were conducted per treatment.

## RNA Extraction and Microarray Analysis

Total RNA was extracted using the nucleospin RNAplant kit (Macherey Nagel, Düren, Germany) and $400~ng$ were amplified using the Ambion messageAmp II (Ambion, Austin, TX, United States) according to the manufacturer's instructions. Five micrograms of amplified RNA were retro-transcribed with $400$ U of Superscript II reverse-transcriptase (Invitrogen Corp., Carlsbad, CA, United States) and labeled with $1.5$ mmol of Cyanine-3 (Cy3) or Cyanine-5 (Cy5) (Interchim, France) and then purified using NucleoSpin Gel and PCR Clean-up column kits (Macherey-Nagel, GmbH and Co., KG, Germany). Purified and labeled cDNA were quantified using a NanoDrop ND-1000. Corresponding Cy3- and Cy5 labeled samples ($30$ pmol) were combined and cohybridized to the Abra_v1.0 12 × 135K arrays on a NimbleGen Hybridization System 4 (mix mode B) at 42°C overnight. The Abra_v1.0 chip was in situ synthesized by Nimblegen (Madison, WI)
and contained 10,633 60-mer oligoprobes that were designed from the *A. brassicicola* automatically annotated genome database<fnref n="4" />, containing one probe per sequence. Slides were washed, dried, and scanned at 2 µm resolution and high sensitivity with a Roche-NimbleGen MS200. DEVA Software version 1.2.1 was used to extract pair data files from the scanned images. Three biological replicates were analyzed per comparison using the dye-switch method. Statistical analyses of the gene expression data were performed using the R language, version 2.14, and the Linear Models for Microarray Analysis package (Smyth et al., 2005) from the Bioconductor project. For the preprocessing step, data were normalized by the LOWESS method. Log ratio and log intensity were calculated before differential expression analyses and performed using the lmFit function and the Bayes-moderated Student's t-test in a linear model for microarray analysis. Probes with P-value <0.05 were considered as differentially expressed. Gene expression datasets were deposited in the Gene Expression Omnibus (GEO)<fnref n="5" />.

## Quantitative PCR

Total RNA was extracted as described above. Amplification experiments were conducted as previously described (Joubert et al., 2011). The relative quantification analysis was performed using the ΔΔCt method (Winer et al., 1999). All the primer sequences used in real-time quantitative PCR are presented in the **Supplementary Table S2**.

## Generation of Targeted Gene Knockout Mutants and Fusion Strains

We followed the procedure described by Pigné et al. (2017). Gene replacement cassettes were generated with the *Hph* gene cassette from pCB1636 (Sweigard et al., 1995) conferring resistance to hygromycin B. The sets of primers used to amplify the 5' and 3' flanking regions of each targeted gene are presented in the **Supplementary Table S2**. The replacement cassettes were used to transform *A. brassicicola* protoplasts as described by Cho et al. (2006). Primer combinations used to confirm integration of the replacement cassette at the targeted locus were presented in **Supplementary Figure S3**.

## Yeast Complementation Assays

Complementation assays were performed in *S. cerevisiae* cells (strains: parental BY4743 Acc#Y20000, ΔHSP12 Acc#Y27070, Euroscarf, Germany) as described by Joubert et al. (2011). Sequences encoding AbSih3 were amplified by PCR with primer pairs AbSih3-Bam (5'-GGATCCAAAAATGACCGACGCATTCCGC-3') - AbSih3-Eco (5'-GAATTCCCATTCCAAGGGCGTTTTTTGG-3'). Unique BamH1-EcoRI restriction sites (indicated in boldface) were thus introduced at the upstream start and downstream of the coding sequences, respectively and were used to clone the PCR product into pGEM-T (Promega) and then into the yeast expression vector pYES2CT (Invitrogen) to generate the

recombinant plasmid pYAbhsp12. Sensitivity to 1 mM H₂O₂ was analyzed based on microcultivation in 300 µl liquid medium. Growth in microplates was automatically recorded using a spectrophotometer (Spectrostar Nano, BMG Labtech) at 30°C.

## Infection Assays

Leaf infection assays were performed on *B. oleracea* plants (var. Bartolo) as described by Joubert et al. (2011). The experiment was repeated twice and involved at least 10 leaves inoculated by each fungal genotype.

Seed infection assays and seed contamination assessments were performed as described by Pochon et al. (2012). At least five plants per fungal genotype were inoculated and the experiment was repeated twice.

## Metabolic Extraction and Amino Acids and Sugar Analysis

Metabolic extractions were made on 15 mg of dry powdered samples. Procedures for methanol-chloroform-water-based extractions as well as determination of amino acid and carbohydrate contents were described by Thouvenot et al. (2015).

## AUTHOR CONTRIBUTIONS

GN'G carried out the sample preparation and microarray analyses. RR, BI, and MM involved in the sample preparation for metabolic analysis and performed the quantitative PCR. NB-S carried out the yeast complementation assays. CM, MM, and CA-B involved in the construction and phenotyping of *Alternaria brassicicola* mutant strains. PS and TG conceived the study and participated in the design of the experiments as well as in the analysis of the results. RL and BP involved in sugar analyses from fungal samples. SP and J-PR involved in the gene expression analyses. CC, MM, FB, BH, AK, and BI involved in the pathological tests.

## FUNDING

This research was supported in part by grants awarded by the Region des Pays de la Loire (FUNHY project). This research was also supported by post-doctoral fellowships (CA-B) provided by the University of Angers.

## ACKNOWLEDGMENTS

The authors thank the Metabolic Profiling and Metabolomic (P2M2) Platform from the French public institute IGEPP for assistance during metabolic phenotyping.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.01969/full#supplementary-material

<footnote n="4">⁴https://genome.jgi.doe.gov/Altbr1/Altbr1.home.html</footnote>

<footnote n="5">⁵https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133507</footnote>
**FIGURE S1** | Comparison of transcript levels obtained by microarray or RT-qPCR methods. The expression of 9 genes was monitored in response to sorbitol and desiccation stresses. The fold inductions (log2 values) are presented in (A) and the correlation plot in (B).

**FIGURE S2** | Total organic contents in a 1 mg sample of dried fungal cultures sample exposed or not to sorbitol and desiccation treatments. (A) Total contents of amino acids. (B) Total content of saccharides and sugar alcohols. Colors display the treatments. Box plots show the median, 1st and 3rd quantiles (n = 3). \*denotes a significant difference (P-value <0.001, Welsh test).

**FIGURE S3** | Generation of null mutants by homologous recombination. (A) Schematic representation of the deleted locus in the wild-type and the

replacement construct with the Hyg B resistance cassette (*Hph* gene). Arrows indicate the position of primers used for the cassette generation and for PCR screening of mutants. (B) Gel electrophoresis of PCR products obtained from template DNA of the wild-type, Δ*absch9*, Δ*absih3* and Δ*absih15* strains with the indicated primer pairs. Molecular sizes (kb) were estimated based on a 1 kb ladder (lane M, NEW ENGLAND BioLabs® Inc.). A supplementary PCR was done with the primer pair ITS1/ITS4 as a positive control to assess the quality of the wild-type template DNA.

<caption>**TABLE S1** | List of predicted sequences from the *Alternaria brassicicola* genome exhibiting physicochemical features of hydrophilins and an obvious match upon Blast analysis with proteins of known functions.</caption>

<caption>**TABLE S2** | List of primers used in this study.</caption>

REFERENCES

Abba' S., Ghignone S., and Bonfante P. (2006). A dehydration-inducible gene in the truffle *Tuber borchii* identifies a novel group of dehydrins. *BMC Genomics* 7:39. doi: 10.1186/1471-2164-7-39

Belmas, E., Briand, M., Kwasiborski, A., Colou, J., N'Guyen, G., Iacomi, B., et al. (2018). Genome sequence of the necrotrophic plant pathogen *Alternaria brassicicola* Abra43. *Genome Announc.* 6:e01559-17. doi: 10.1128/genomeA.01559-17

Brewster, J. L., and Gustin, M. C. (2014). Hog1: 20 years of discovery and impact. *Sci. Signal.* 7:re7. doi: 10.1126/scisignal.2005458

Calahan, D., Dunham, M., DeSevo, C., and Koshland, D. E. (2011). Genetic analysis of desiccation tolerance in *Sachcharomyces cerevisiae*. *Genetics* 189, 507–519. doi: 10.1534/genetics.111.130369

Carrere, S., and Gouzy, J. (2017). myGenomeBrowser: building and sharing your own genome browser. *Bioinformatics* 33, 1255–1257. doi: 10.1093/bioinformatics/btw800

Cho, Y., Davis, J. W., Kim, K.-H., Wang, J., Sun, Q.-H., Cramer, R. A. Jr., et al. (2006). A high throughput targeted gene disruption method for *Alternaria brassicicola* functional genomics using linear minimal element (LME) constructs. *Mol. Plant Microbe Interact.* 19, 7–15. doi: 10.1094/MPMI-19-0007

Dang, H. X., Pryor, B., Peever, T., and Lawrence, C. B. (2015). The *Alternaria* genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species. *BMC Genomics* 16:239. doi: 10.1186/s12864-015-1430-7

Dang, N. X., and Hincha, D. K. (2011). Identification of two hydrophilins that contribute to the desiccation and freezing tolerance of yeast (*Saccharomyces cerevisiae*) cells. *Cryobiology* 62, 188–193. doi: 10.1016/j.cryobiol.2011.03.002

de Lima Alves, F., Stevenson, A., Baxter, E., Gillion, J. L., Hejazi, F., Hayes, S., et al. (2015). Concomitant osmotic and chaotropicity-induced stresses in *Aspergillus wentii*: compatible solutes determine the biotic window. *Curr. Genet.* 61, 457–477. doi: 10.1007/s00294-015-0496-8

Dixon, K. P., Xu, J. R., Smirnoff, N., and Talbot, N. J. (1999). Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by *Magnaporthe grisea*. *Plant Cell* 11, 2045–2058. doi: 10.1105/tpc.11.10.2045

Dongo, A., Bataillé-Simoneau, N., Campion, C., Guillemette, T., Hamon, B., Iacomi-Vasilescu, B., et al. (2009). The group III two-component histidine kinase of filamentous fungi is involved in the fungicidal activity of the bacterial polyketide ambruticin. *Appl. Environ. Microbiol.* 75, 127–134. doi: 10.1128/AEM.00993-08

Dupont, S., Rapoport, A., Gervais, P., and Beney, L. (2014). Survival kit of *Saccharomyces cerevisiae* for anhydrobiosis. *Appl. Microbiol. Biotechnol.* 98, 8821–8834. doi: 10.1007/s00253-014-6028-5

Gadd, G. M., Chalmers, K., and Reed, R. H. (1987). The role of trehalose in dehydration resistance of *Saccharomyces cerevisiae*. *FEMS Microbiol. Lett.* 48, 249–254. doi: 10.1111/j.1574-6968.1987.tb02551.x

Garay-Arroyo, A., Colmenero-Flores, J. M., Garciarrubio, A., and Covarrubias, A. A. (2000). Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. *J. Biol. Chem.* 275, 5668–5674. doi: 10.1074/jbc.275.8.5668

Gaucher, M., Dugé de Bernonville, T., Lohou, D., Guyot, S., Guillemette, T., Brisset, M.-N., et al. (2013). Histolocalization and physico-chemical characterization of dihydrochalcones: insight into the role of apple major flavonoids. *Phytochemistry* 90, 78–89. doi: 10.1016/j.phytochem.2013.02.009

Goyal, K., Walton, L. J., and Tunnacliffe, A. (2005). LEA proteins prevent protein aggregation due to water stress. *Biochem. J.* 388, 151–157. doi: 10.1042/BJ20041931

Gu, Q., Zhang, C., Yu, F., Yin, Y., Shim, W.-B., and Ma, Z. (2015). Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in *Fusarium graminearum*. *Environ. Microbiol.* 17, 2661–2676. doi: 10.1111/1462-2920.12522

Hildebrandt, T. M., Nunes Nesi, A., Araújo, W. L., and Braun, H.-P. (2015). Amino acid catabolism in plants. *Mol. Plant* 8, 1563–1579. doi: 10.1016/j.molp.2015.09.005

Hohmann, S. (2015). An integrated view on a eukaryotic osmoregulation system. *Curr. Genet.* 61, 373–382. doi: 10.1007/s00294-015-0475-0

Huber, A., Bodenmiller, B., Uotila, A., Stahl, M., Wanka, S., Gerrits, B., et al. (2009). Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. *Genes Dev.* 23, 1929–1943. doi: 10.1101/gad.532109

Iacomi-Vasilescu, B., Bataille-Simoneau, N., Campion, C., Dongo, A., Laurent, E., Serandat, I., et al. (2008). Effect of null mutations in the *AbNIK1* gene on saprophytic and parasitic fitness of *Alternaria brassicicola* isolates highly resistant to dicarboximide fungicides. *Plant Pathol.* 57, 937–947. doi: 10.1111/j.1365-3059.2008.01864.x

Joubert, A., Bataille-Simoneau, N., Campion, C., Guillemette, T., Hudhomme, P., Iacomi-Vasilescu, B., et al. (2011). Cell wall integrity and high osmolarity glycerol pathways are required for adaptation of *Alternaria brassicicola* to cell wall stress caused by brassicaceous indolic phytoalexins. *Cell. Microbiol.* 13, 62–80. doi: 10.1111/j.1462-5822.2010.01520.x

Joubert, A., Calmes, B., Berruyer, R., Pihet, M., Bouchara, J.-P., Simoneau, P., et al. (2010). Laser nephelometry applied in an automated microplate system to study filamentous fungus growth. *Biotechniques* 48, 399–404. doi: 10.2144/000113399

Kayingo, G., and Wong, B. (2005). The MAP kinase Hog1p differentially regulates stress-induced production and accumulation of glycerol and D-arabinol in *Candida albicans*. *Microbiology* 151, 2987–2999. doi: 10.1099/mic.0.28040-0

López-Martínez, G., Rodríguez-Porrata, B., Margalef-Català, M., and Cordero-Otero, R. (2012). The STF2p hydrophilin from *Saccharomyces cerevisiae* is required for dehydration stress tolerance. *PLoS One* 7:e33324. doi: 10.1371/journal.pone.0033324

Lowe, R. G. T., Lord, M., Rybak, K., Trengove, R. D., Oliver, R. P., and Solomon, P. S. (2008). A metabolomic approach to dissecting osmotic stress in the wheat pathogen *Stagonospora nodorum*. *Fungal Genet. Biol.* 45, 1479–1486. doi: 10.1016/j.fgb.2008.08.006

Ohm, R. A., Feau, N., Henrissat, B., Schoch, C. L., Horwitz, B. A., Barry, K. W., et al. (2012). Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi. *PLoS Pathog.* 8:e1003037. doi: 10.1371/journal.ppat.1003037

Pascual, S., Melgarejo, P., and Magan, N. (2003). Water availability affects the growth, accumulation of compatible solutes and the viability of the biocontrol agent *Epicoccum nigrum*. *Mycopathologia* 156, 93–100.
Pascual-Ahuir, A., and Proft, M. (2007). The Sch9 kinase is a chromatin-associated transcriptional activator of osmostress-responsive genes. *EMBO J.* 26, 3098–3108. doi: 10.1038/sj.emboj.7601756

Pigné, S., Zykwinska, A., Janod, E., Cuenot, S., Kerkoud, M., Raulo, R., et al. (2017). A flavoprotein supports cell wall properties in the necrotrophic fungus *Alternaria brassicicola*. *Fungal Biol. Biotechnol.* 4:1. doi: 10.1186/s40694-016-0029-3

Pochon, S., Simoneau, P., Pigné, S., Balidas, S., Bataillé-Simoneau, N., Campion, C., et al. (2013). Dehydrin-like proteins in the necrotrophic fungus *Alternaria brassicicola* have a role in plant pathogenesis and stress response. *PLoS One* 8:e75143. doi: 10.1371/journal.pone.0075143

Pochon, S., Terrasson, E., Guillemette, T., Iacomi-Vasilescu, B., Georgeault, S., Juchaux, M., et al. (2012). The *Arabidopsis thaliana-Alternaria brassicicola* pathosystem: a model interaction for investigating seed transmission of necrotrophic fungi. *Plant Methods* 8:16. doi: 10.1186/1746-4811-8-16

Ravishankar, J. P., Suryanarayanan, T. S., and Muruganandam, V. (2006). Strategies for osmoregulation in the marine fungus *Cirrenalia pygmea* Kohl.(Hyphomycetes). *Indian J. Geo Mar. Sci.* 35, 351–358.

Reyes, J. L., Rodrigo, M.-J., Colmenero-Flores, J. M., Gil, J.-V., Garay-Arroyo, A., Campos, F., et al. (2005). Hydrophilins from distant organisms can protect enzymatic activities from water limitation effects in vitro. *Plant Cell Environ.* 28, 709–718. doi: 10.1111/j.1365-3040.2005.01317.x

Rodríguez-Porrata, B., Carmona-Gutierrez, D., Reisenbichler, A., Bauer, M., Lopez, G., Escoté, X., et al. (2012). Sip18 hydrophilin prevents yeast cell death during desiccation stress. *J. Appl. Microbiol.* 112, 512–525. doi: 10.1111/j.1365-2672. 2011.05219.x

Sales, K., Brandt, W., Rumbak, E., and Lindsey, G. (2000). The LEA-like protein HSP 12 in *Saccharomyces cerevisiae* has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress. *Biochim. Biophys. Acta* 1463, 267–278. doi: 10.1016/s0005-2736(99)00215-1

Sánchez-Fresneda, R., Guirao-Abad, J. P., Argüelles, A., González-Párraga, P., Valentín, E., and Argüelles, J.-C. (2013). Specific stress-induced storage of trehalose, glycerol and d-arabitol in response to oxidative and osmotic stress in *Candida albicans*. *Biochem. Biophys. Res. Commun.* 430, 1334–1339. doi: 10.1016/j.bbrc.2012.10.118

Sellam, A., Iacomi-Vasilescu, B., Hudhomme, P., and Simoneau, P. (2007). In vitro antifungal activity of brassinin, camalexin and two isothiocyanates against the crucifer pathogens *Alternaria brassicicola* and *Alternaria brassicaceae*. *Plant Pathol.* 56, 296–301. doi: 10.1111/j.1365-3059.2006.01497.x

Smyth, G. K., Michaud, J., and Scott, H. S. (2005). Use of within-array replicate spots for assessing differential expression in microarray experiments. *Bioinformatics* 21, 2067–2075. doi: 10.1093/bioinformatics/bti270

Soanes, D. M., Alam, I., Cornell, M., Wong, H. M., Hedeler, C., Paton, N. W., et al. (2008). Comparative genome analysis of filamentous fungi reveals gene family expansions associated with fungal pathogenesis. *PLoS One* 3:e2300. doi: 10.1371/journal.pone.0002300

Sweigard, J. A., Carroll, A. M., Kang, S., Farrall, L., Chumley, F. G., and Valent, B. (1995). Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. *Plant Cell* 7, 1221–1233. doi: 10.1105/tpc.7.8.1221

Tapia, H., and Koshland, D. E. (2014). Trehalose is a versatile and long-lived chaperone for desiccation tolerance. *Curr. Biol.* 24, 2758–2766. doi: 10.1016/j.cub.2014.10.005

Thouvenot, L., Deleu, C., Berardocco, S., Haury, J., and Thiébaut, G. (2015). Characterization of the salt stress vulnerability of three invasive freshwater plant species using a metabolic profiling approach. *J. Plant Physiol.* 175, 113–121. doi: 10.1016/j.jplph.2014.11.007

Tunnacliffe, A., and Wise, M. J. (2007). The continuing conundrum of the LEA proteins. *Naturwissenschaften* 94, 791–812. doi: 10.1007/s00114-007-0254-y

van den Bosch, F., Fraaije, B. A., van den Berg, F., and Shaw, M. W. (2010). Evolutionary bi-stability in pathogen transmission mode. *Proc. Biol. Sci.* 277, 1735–1742. doi: 10.1098/rspb.2009.2211

Wang, S., Liu, Y., and Xu, J. (2017). Comparison of different drying methods for recovery of mushroom DNA. *Sci. Rep.* 7:3008. doi: 10.1038/s41598-017-03570-7

Wartenberg, D., Vödisch, M., Kniemeyer, O., Albrecht-Eckardt, D., Scherlach, K., Winkler, R., et al. (2012). Proteome analysis of the farnesol-induced stress response in *Aspergillus nidulans*. The role of a putative dehydrin. *J. Proteomics* 75, 4038–4049. doi: 10.1016/j.jprot.2012.05.023

Winer, J., Jung, C. K., Shackel, I., and Williams, P. M. (1999). Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes *in vitro*. *Anal. Biochem.* 270, 41–49. doi: 10.1006/abio.1999.4085

Wong Sak Hoi, J., Beau, R., and Latgé, J.-P. (2012). A novel dehydrin-like protein from *Aspergillus fumigatus* regulates freezing tolerance. *Fungal Genet. Biol.* 49, 210–216. doi: 10.1016/j.fgb.2012.01.005

Wong Sak Hoi, J., Lamarre, C., Beau, R., Meneau, I., Berepiki, A., Barre, A., et al. (2011). A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen *Aspergillus fumigatus*. *Mol. Biol. Cell* 22, 1896–1906. doi: 10.1091/mbc.E10-11-0914

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 N'Guyen, Raulo, Marchi, Agustí-Brisach, Iacomi, Pelletier, Renou, Bataillé-Simoneau, Campion, Bastide, Hamon, Mouchès, Porcheron, Lemoine, Kwasiborski, Simoneau and Guillemette. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
# Advantages of publishing in Frontiers

<figure><img src="image_80.png" /></figure>

**OPEN ACCESS**

Articles are free to read for greatest visibility and readership

<figure><img src="image_81.png" /></figure>

**FAST PUBLICATION**

Around 90 days from submission to decision

<figure><img src="image_82.png" /></figure>

**HIGH QUALITY PEER-REVIEW**

Rigorous, collaborative, and constructive peer-review

<figure><img src="image_83.png" /></figure>

**TRANSPARENT PEER-REVIEW**

Editors and reviewers acknowledged by name on published articles

**Frontiers**

Avenue du Tribunal-Fédéral 34
1005 Lausanne | Switzerland

Visit us: www.frontiersin.org

Contact us: info@frontiersin.org | +41 21 510 17 00

<figure><img src="image_84.png" /></figure>

**REPRODUCIBILITY OF RESEARCH**

Support open data and methods to enhance research reproducibility

<figure><img src="image_85.png" /></figure>

**DIGITAL PUBLISHING**

Articles designed for optimal readership across devices

<figure><img src="image_86.png" /></figure>

**FOLLOW US**

@frontiersin

<figure><img src="image_87.png" /></figure>

**IMPACT METRICS**

Advanced article metrics track visibility across digital media

<figure><img src="image_88.png" /></figure>

**EXTENSIVE PROMOTION**

Marketing and promotion of impactful research

<figure><img src="image_89.png" /></figure>

**LOOP RESEARCH NETWORK**

Our network increases your article's readership