Functional genomics identifies a small secreted protein that plays a role during the biotrophic to necrotrophic shift in the root rot pathogen Phytophthora medicaginis

Abstract

Introduction: Hemibiotrophic Phytophthora are a group of agriculturally and ecologically important pathogenic oomycetes causing severe decline in plant growth and fitness. The lifestyle of these pathogens consists of an initial biotrophic phase followed by a switch to a necrotrophic phase in the latter stages of infection. Between these two phases is the biotrophic to necrotrophic switch (BNS) phase, the timing and controls of which are not well understood particularly in Phytophthora spp. where host resistance has a purely quantitative genetic basis.

Methods: To investigate this we sequenced and annotated the genome of Phytophthora medicaginis, causal agent of root rot and substantial yield losses to Fabaceae hosts. We analyzed the transcriptome of P. medicaginis across three phases of colonization of a susceptible chickpea host (Cicer arietinum) and performed co-regulatory analysis to identify putative small secreted protein (SSP) effectors that influence timing of the BNS in a quantitative pathosystem.

Results: The genome of P. medicaginis is ~78 Mb, comparable to P. fragariae and P. rubi which also cause root rot. Despite this, it encodes the second smallest number of RxLR (arginine-any amino acid-leucine-arginine) containing proteins of currently sequenced Phytophthora species. Only quantitative resistance is known in chickpea to P. medicaginis, however, we found that many RxLR, Crinkler (CRN), and Nep1-like protein (NLP) proteins and carbohydrate active enzymes (CAZymes) were regulated during infection. Characterization of one of these, Phytmed_10271, which encodes an RxLR effector demonstrates that it plays a role in the timing of the BNS phase and root cell death.

Discussion: These findings provide an important framework and resource for understanding the role of pathogenicity factors in purely quantitative Phytophthora pathosystems and their implications to the timing of the BNS phase.

Keywords: co-expression network; effector; genome; hemibiotrophic pathogenesis; immune manipulation.

Figure 1

Phytophthora medicaginis displays phase-specific Gene Ontology (GO) biological processes during chickpea root infection. (A) Venn diagram showing common and unique P. medicaginis up-regulated genes at 12-, 24- and 72-hours post inoculation. (B) Venn diagram showing common and unique P. medicaginis down-regulated gene sets at 12-, 24- and 72-hours post inoculation. (C) Enriched GO biological processes associated with combined uniquely up- and down-regulated genes in P. medicaginis at the three stages of hemibiotrophic infection. The GO terms are shown on the y-axes. The number of genes associated with each GO term are shown on the x-axes. The Fisher’s exact test p-value statistic for each GO term is shown based on a blue-yellow gradient (p < 0.05). (D) Mean intensity of reactive oxygen species (ROS) staining in root compartment. (E) Mean intensity of ROS staining in hyphae. + SE. The x-axis shows the time points post inoculation in hours and the y-axis shows the mean intensity of ROS staining. BP, biotrophic phase; BNS, biotrophic to necrotrophic switch phase; NP, necrotrophic phase; hpi, hours post inoculation.

Figure 2

Putative P. medicaginis small secreted proteins (SSPs) display phase specific regulation during the lifestyle switch of P. medicaginis. (A) Identification of SSPs in the genome of P. medicaginis using SignalP (Almagro Armenteros et al., 2019) and TMHMM (Krogh et al., 2001) software and filtering for amino acid size < 300 amino acids using a python script. (B) Heatmap of 292 SSPs regulated at 12-, 24- and 72-hours post inoculation encompassing the biotrophic, switch and necrotrophic phases, respectively. (C) Venn diagram of significantly differentially regulated common and unique SSPs at the three phases of hemibiotrophic infection (p < 0.05). BP, biotrophic phase; BNS, biotrophic to necrotrophic switch phase; NP, necrotrophic phase; hpi, hours post inoculation.

Figure 3

Putative Phytophthora medicaginis small secreted protein (SSP) Phytmed_10271 displays co-regulation with chickpea defense pathways. (A) Enriched Gene Ontology biological processes associated with chickpea genes co-expressed with Phytmed_1027. The GO terms are shown on the y-axis. The number of genes associated with each GO term are shown on the x-axis. The Fisher’s exact test p-value statistic for each GO term is shown based on a blue-yellow gradient (p < 0.05). (B) Annotated Phytmed_10271 protein with motifs present at specific regions within the amino acid sequence. (C) Fold change in expression of Phytmed_10271 relative to control at three phases of hemibiotrophic infection. BP, biotrophic phase; BNS, biotrophic to necrotrophic switch phase; NP, necrotrophic phase; hpi, hours post inoculation. The bars indicate standard error.

Figure 4

The Phytmed_10271 knockdown displays a higher rate of tissue necrosis at the biotrophic to necrotrophic switch phase. (A) Symptoms in chickpea var. ‘Sonali’ roots, inoculated with P. medicaginis and either sprayed with dsiRNA targeting Phytmed_10271 (Phytmed_10271 dsiRNA), sterile water (RNA-negative control) or scrambled dsiRNA not targeting any gene (control) at 24 hpi, the BNS phase." Here AND is replaced with OR. A representative photo of a root for each treatment with the inoculated site centered in the middle of the root are shown. (B) Electrolyte leakage cell death analysis of chickpea var. ‘Sonali’ roots at 24 hpi, the BNS phase. The x-axis shows the treatments including P. medicaginis inoculated roots sprayed with Phytmed_10271 dsiRNA, sterile water and scrambled dsiRNA. The y-axis shows the percentage of electrolyte leakage of pre-boiled relative to post-boiled root samples. Lower case letters indicate significant differences (ANOVA Tukey method). (C) Hyphal extension analysis of Phytmed_10271 dsiRNA, RNA-negative control and scrambled dsiRNA treatments (ANOVA and Tukey post-hoc test). (D) Electrolyte leakage analysis on roots from plant only set up sprayed with either dsiRNA targeting Phytmed_10271, scrambled dsiRNA or sterile water (ANOVA and Tukey test). (E) Phytmed_10271 displays 3x less abundance in the Phytmed_10271 dsiRNA knockdown treatment compared to scrambled dsiRNA. Data shown are fold change of normalized unique counts in Phytmed_10271 dsiRNA relative to the scrambled dsiRNA. (F) The Phytmed_10271 SSP and chickpea gene co-expression network. The red node indicates the Phytmed_10271 SSP, grey nodes indicate chickpea genes, and black nodes indicate the 73 significantly differentially regulated chickpea genes in the Phytmed_10271 dsiRNA knockdown relative to the scrambled dsiRNA control (Log₂FC > 1 and < -1, p < 0.05). The strength of the edges connecting nodes are shown by a color gradient (Spearman rank: -1 to 1 as yellow to purple). Dotted lines represent negative correlations and solid lines represent positive correlations. (G) Hierarchical clustering of the significantly differentially regulated chickpea genes in the Phytmed_10271 dsiRNA knockdown relative to the scrambled dsiRNA control (Log₂FC > 1 and < -1, p < 0.05). Examples of genes present within the network that were significantly differentially regulated by knockdown of Phytmed_10271 are highlighted by arrows including IAA26, NDOLE-3-ACETIC ACID INDUCIBLE 26 Protein Phosphatase 2C; CML27, Calcium-binding protein 27; MLP-like 43, Major Latex Protein-like 43; TIP, Tonoplast Intrinsic Protein and FMO, Flavin-binding monooxygenase family protein. BNS, biotrophic to necrotrophic switch; hpi, hours post inoculation; SSP, small secreted protein.