Chickpea Roots Undergoing Colonisation by Phytophthora medicaginis Exhibit Opposing Jasmonic Acid and Salicylic Acid Accumulation and Signalling Profiles to Leaf Hemibiotrophic Models

Abstract

Hemibiotrophic pathogens cause significant losses within agriculture, threatening the sustainability of food systems globally. These microbes colonise plant tissues in three phases: a biotrophic phase followed by a biotrophic-to-necrotrophic switch phase and ending with necrotrophy. Each of these phases is characterized by both common and discrete host transcriptional responses. Plant hormones play an important role in these phases, with foliar models showing that salicylic acid accumulates during the biotrophic phase and jasmonic acid/ethylene responses occur during the necrotrophic phase. The appropriateness of this model to plant roots has been challenged in recent years. The need to understand root responses to hemibiotrophic pathogens of agronomic importance necessitates further research. In this study, using the root hemibiotroph Phytophthora medicaginis, we define the duration of each phase of pathogenesis in Cicer arietinum (chickpea) roots. Using transcriptional profiling, we demonstrate that susceptible chickpea roots display some similarities in response to disease progression as previously documented in leaf plant-pathogen hemibiotrophic interactions. However, our transcriptomic results also show that chickpea roots do not conform to the phytohormone responses typically found in leaf colonisation by hemibiotrophs. We found that quantified levels of salicylic acid concentrations in root tissues decreased significantly during biotrophy while jasmonic acid concentrations were significantly induced. This study demonstrated that a wider spectrum of plant species should be investigated in the future to understand the physiological changes in plants during colonisation by soil-borne hemibiotrophic pathogens before we can better manage these economically important microbes.

Keywords: Cicer arietinum; disease development; pathogenesis; phytohormone; plant immunity; transcriptomics.

Figure 1

Phytophthora medicaginis pathogenesis is associated with a fast rate of necrosis and hemibiotrophic development. (a–d) Macroscopic symptoms of chickpea var. ‘Sonali’ either mock-inoculated with pathogen free agar blocks (control) or 12, 24 and 72 h post-inoculation (hpi) with P. medicaginis isolate 7831. (e–h) Confocal microscopy images of the 0.5 cm root segment that was sampled either from a mock-inoculated control or from the P. medicaginis inoculated site at 12, 24 and 72 hpi (sampled segment denoted by a black box). P. medicaginis hyphae are observed by green fluorescence while root cell walls are observed by red fluorescence. Intracellular haustoria development are indicated with white arrows while intracellular and extracellular chlamydospores are indicated by blue arrows. (i) Confocal microscopy images showing close up of P. medicaginis haustoria (white arrows) and (j) chlamydospores (blue arrows) photographed at 12 and 72 hpi, respectively. Scale bars: (e,f) 150 µm; (g,h) 1100 µm; (i) 70 µm; (j) 60 µm. The confocal microscopy images were taken using a TCS SP5 confocal laser scanning microscope. For imaging, a Z-stack was taken of the inoculated site using whole-roots roots for (e, h–j), whereas 30 µm in thickness longitudinal sections of cortical cell layer were imaged at the inoculated site for (f,g).

Figure 2

Phytophthora medicaginis displays a short biotrophic phase up to 24 h post-inoculation (hpi) before switching to a necrotrophic phase. (a) P. medicaginis induces significant root cell death from 36 hpi onwards. The x-axis shows the timepoints post-inoculation in hours (h) and the y-axis shows the arcsine-transformed electrolyte leakage proportions of pre-boiled relative to post-boiled root samples. * = significant differences in arcsine-transformed electrolyte leakage proportions between the inoculated and mock-inoculated control roots at a specific timepoints (p < 0.001, LSD = 0.08). (b) P. medicaginis causes significantly lower root cell viability at 72 hpi. The x-axis shows the control and timepoints post-inoculation in hours (h), and the y-axis shows the ratio of living to dead cells. Lower-case letters indicate significant difference between treatments (ANOVA, p < 0.05; n = 6).

Figure 3

Genome-wide evaluation of RNA-sequencing data in chickpea var. ‘Sonali’ roots undergoing colonisation by Phytophthora medicaginis. (a) Principal component analysis of RNA-seq samples at 12, 24 and 72 h post-inoculation (hpi), and mock-inoculated control. (b) Number of significantly up-regulated and down-regulated genes in chickpea var. ‘Sonali’ roots at 12, 24 and 72 hpi (p < 0.05, 1 < log2FC > −1). BP: biotrophic phase, BNS: biotrophy to necrotrophy switch and NP: necrotrophic phase.

Figure 4

Clusters of significantly differentially regulated chickpea genes display conserved expression patterns across three phases of Phytophthora medicaginis colonisation. Hierarchical clustering of the log2-transformed data of 13,568 significantly regulated genes of chickpea variety ‘Sonali’ during the three phases of colonisation by P. medicaginis are represented (padj-value < 0.05, 1 < log2FC > −1). All data points are the ratio of transcript abundance in colonised roots as compared to control plants grown under the same conditions where red value indicates increased gene expression and black/grey indicates decreased gene expression. The heat map is annotated on the left-hand side with the hierarchical cluster groupings denoted i–xi. BP: biotrophic phase, BNS: biotrophy to necrotrophy switch, hpi: hours post-inoculation, NBS-LRR: nucleotide-binding site-leucine-rich repeat and NP: necrotrophic phase.

Figure 5

Chickpea unique responses at each of the phases reveal similar and different responses observed in other models. (a) Venn diagrams showing the number of common and unique significantly regulated genes in chickpea var. ‘Sonali’ varies during three timepoints of Phytophthora medicaginis colonisation (12, 24, and 72 h post-inoculation (hpi)), pertaining to the biotrophic phases (BP), biotrophy to necrotrophy switch (BNS) phase and necrotrophic phase (NP), respectively. The red and black Venn diagrams show the common and unique genes pertaining to the significantly up-regulated and down-regulated gene sets, respectively. The white, brown and gold circles pertain to the uniquely regulated genes that were used for gene ontology (GO) enrichment analysis at a specific phase of infection. (b) Enriched GO biological processes associated with differentially expressed genes in Sonali roots unique to each of the three phases of P. medicaginis pathogenesis. The GO terms associated with each timepoint are shown on the Y axes and 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 are shown based on a blue–yellow gradient. GO terms discussed within the text are highlighted in orange.

Figure 6

Jasmonic acid (JA), ethylene (ET), and salycilic acid (SA) signalling pathways in chickpea roots during the biotrophic to necrotrophic switch of Phytophthora medicaginis. (a) Expression profiles of the log2-transformed data of differentially regulated genes pertaining to the JA signalling pathways in chickpea var. ‘Sonali’ at 12, 24 and 72 h post-inoculation (hpi). Genes are annotated with Arabidopsis orthologue: LOX, lipoxygenase (Biosynthesis); AOS, allene oxide synthase (Biosynthesis); JAZ, jasmonate zim domain protein (Perception); DEF, defensin-like gene (Signalling); PR3, pathogenesis-related 3 (Signalling); PR4, pathogenesis-related 4 (Signalling). These latter two genes are also involved in ET signalling. (b) Expression profiles of the log2-transformed data of differentially regulated genes pertaining to the ET pathway in chickpea var. ‘Sonali’ at 12, 24, 72 hpi. Genes are annotated with the Arabidopsis orthologue: ACS, acc synthase (Biosynthesis); ACO, acc oxidase (Biosynthesis); ERF9, ethylene response factor 9 (Signalling); EIN3, ethylene insensitive 3 (Signalling); ERF1, ethylene response factor 1 (Ssignalling). (c) Expression profiles of the log2-transformed data of differentially regulated genes pertaining to the SA signalling pathways in chickpea variety ‘Sonali’ at 12, 24 and 72 hpi. Down-regulated genes and up-regulated genes are presented as black (low) or red (high), respectively. Genes are annotated with Arabidopsis orthologue: EDS5, enhanced disease susceptibility 5 (Biosynthesis); ICS, isochorismate synthase (Biosynthesis); NPR1, nonexpressor of pathogenesis-related 1 (Perception); WRKY70, wall-associated receptor kinase 70 (Signalling); TGA4, tcagc-binding factor 4 (Signalling); PR1, pathogenesis-related 1 (Signalling). (d) Percent change in hormone concentrations in roots undergoing colonisation by P. medicaginis (compared to axenically grown control roots) for JA (orange solid line), JA-Ile (orange dashed line), and SA (black solid line). The error bars indicate standard error between 9–12 biological replicates at each timepoint; * = Significant difference from control values (p < 0.05; Student’s T-test). BP: biotrophic phase, BNS: biotrophy to necrotrophy switch, NP: necrotrophic phase.