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

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 analysed the transcriptome of P. medicaginis across three phases of colonisation 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. Characterisation 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. Overall design: In total six samples were generated for the double stranded interfering RNA knockdown experiment to test the function of Phytmed_10271 in Phytophthora medicaginis during their biotrophic to necrotrophic switch in chickpea roots. Three of the samples were from dsiRNA treated roots targeting Phytmed_10271 and the other three samples were from roots treated with scrambled dsiRNA unable to target any endogenous P. medicaginis genes. Additionally, 16 samples were generated for RNA-sequencing of which the 16 samples included 1 replicate each of 16 timepoints 8, 16, 20, 28, 32, 36, 40, 44, 48, 60, 84, 96, 108, 120, 132 and 144 hours post P. medicaginis inoculation in chickpea var. 'Sonali' roots.