pH effect on strain-specific transcriptomes of the take-all fungus

The soilborne fungus Gaeumannomyces tritici (G. tritici) causes the take-all disease on wheat roots. Ambient pH has been shown to be critical in different steps of G. tritici life cycle such as survival in bulk soil, saprophytic growth, and pathogenicity on plants. There are however intra-specific variations and we previously found two types of G. tritici strains that grow preferentially either at acidic pH or at neutral/alkaline pH; gene expression involved in pH-signal transduction pathway and pathogenesis was differentially regulated in two strains representative of these types. To go deeper in the description of the genetic pathways and the understanding of this adaptative mechanism, transcriptome sequencing was achieved on two strains (PG6 and PG38) which displayed opposite growth profiles in two pH conditions (acidic and neutral). PG6, growing better at acidic pH, overexpressed in this condition genes related to cell proliferation. In contrast, PG38, which grew better at neutral pH, overexpressed in this condition genes involved in fatty acids and amino acid metabolisms, and genes potentially related to pathogenesis. This strain also expressed stress resistance mechanisms at both pH, to assert a convenient growth under various ambient pH conditions. These differences in metabolic pathway expression between strains at different pH might buffer the effect of field or soil variation in wheat fields, and explain the success of the pathogen.


INTRODUCTION
0.22 µm pore-size sterile membrane (Millipore, Molsheim, France) was laid on the agar 110 surface to force mycelium to grow on top of the medium and to facilitate its sampling for 111 further RNA extractions. Five mm diameter plugs of mycelium were removed from the edge 112 of a colony grown twice the non-buffered medium. One plug per plate was laid on buffered 113 Fåhraeus media (A or N), in the centre of Petri dishes, and the plates, covered by the 114 polycarbonate filter, were incubated for 7 days at 20°C in the dark. Two orthogonal diameters 115 of the colony were measured in each condition 7 days after inoculation. For each pH 116 condition and strain, three plates were used, and three independent experiments were 117 performed. Mycelium growth was compared using the Wilcoxon rank sum test.  Read mapping to the reference genome and transcript counting 143 STAR v2.5.2a_modified [17] was used to align the reads to the published genome of Ggt 144 strain (https://fungi.ensembl.org/Gaeumannomyces_graminis/Info/Index). This available 145 reference sequenced genome is from the R3-111a-1 Ggt strain, different from the strains used 146 in our study [15,18]   The following parameters were applied: 5 min at 25°C, 1 h at 42°C and 15 min at 70°C.

181
Reactions without RNA or without reverse transcriptase were performed as negative controls.

182
The oligonucleotides designed with the Primer 3 software are described in S2 Table. 183 Quantitative PCR reactions (20 µL) containing 1 µl of cDNA, 0.4 µM of each primer and 1 X  Table). A dissociation stage was applied at the end of the PCR to assess that each 188 amplicon generated was specific. Moreover, each specific amplicon was sequenced  The two Ggt strains (PG6 and PG38) were grown on media buffered at pH 4.6 (A) or 7.0 198 (N) in the dark. After 7 days, two orthogonal diameters of the colony were measured in each 199 condition (Fig 1). Both strains grow at both pH levels but displayed two different growth

242
The percentages are given from initial raw reads. Validation and clustering of the RNA-Seq data 248 We looked for differential gene expression between the different samples and normalized

249
DESeq2 counts of all samples were plotted on a PCA to estimate the variability of the 250 experiments and the biological conditions (Fig 2). 86% of the variance was represented on the 251 plot. We confirmed that the three replicates of each experimental condition were largely 252 clustered together, which validate our RNA-Seq experiment. The x-axis (57%) separated 253 clearly the two strains whereas the y-axis (29%) was representative of the pH effect. biological condition (Fig. 3). Expression profiles were clearly different between conditions, 262 identifying groups of up-regulated (red) and down-regulated (green) genes. Gene expression 263 was affected by the pH and/or by the strain. A clear separation was seen first between the 264 strains, and secondly between pH inside each strain. Gene expression levels were displayed from green (downregulated) to red (upregulated). Colored bars on the left 269 of the heatmap mark distinct major branches in the clustering tree grouping genes with similar expression pattern.

270
Each row corresponded to a single gene and each column to the mean of the three biological repetitions of one "gplots" R package.   The UpSetR package was used to identify 798 genes specifically regulated between 331 acidic and neutral pH whatever the strain (black long arrows on Fig. 5 and Table 3), and 332 1,267 genes regulated between the two strains independently of the pH (grey long arrows on 333 Fig. 5 and Table 3).

361
Enrichment factor (> 5) was represented on the x-axis and enriched GO-terms were described on the y-axis. signaling pathway and could, for example, explain a potential ability of this strain to resist to 377 antibiotics synthetized by antagonists microogranisms in soils [6]. So whatever the pH, the 378 PG38 seemed to apply DNA repair and stress resistance mechanisms which could be essential   terms were enriched in neutral conditions (Fig. 6C) whatever the strain and 9 in acidic 398 conditions (Fig. 6D). 399 Several enriched GO-terms in neutral conditions were linked to 'V-ATPases' and 'proton between neutral and acidic conditions. So both strains of our study seemed to be able, at the 406 same level, to maintain pH-gradients in their cells and organelles when they grew on neutral 407 medium. Enrichment of 'ascospore wall assembly' at neutral pH was also seen, suggesting an 408 ability of both strains at this pH to form ascospores. As this ability is related to the sexual 409 stage of Ggt, which is usually used to disperse at long distance or to resist to stresses, it could pathway. At acidic pH, despite a lower growth, this strain displayed ability to stress resistance 506 potentially linked to survivability. In total, whatever the pH but particularly at neutral pH,

507
PG38 could have better abilities to survive in soils during intercrops, so allowing the presence 508 of inoculum source to the next wheat culture. Thus, we described, in an original way, 509 different pH-dependent strategies related to growth rate profiles in the two studied strains.