Table5_Functional genomics identifies a small secreted protein that plays a role during the biotrophic to necrotrophic shift in the root rot pathogen Phytophthora medicaginis.xlsx

IntroductionHemibiotrophic 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.
MethodsTo 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.
ResultsThe 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.
DiscussionThese 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.

引言
半活体营养型疫霉（Hemibiotrophic Phytophthora）是一类兼具农业与生态重要性的致病性卵菌，可导致植物生长与健康状况严重衰退。该类病原菌的生活史包含初始的活体营养阶段，随后在侵染后期切换为死体营养阶段。介于二者之间的是活体营养向死体营养转换（Biotrophic to Necrotrophic Switch, BNS）阶段，其发生时机与调控机制目前尚未得到充分阐明，尤其在寄主抗性仅具备数量遗传基础的疫霉属（Phytophthora spp.）物种中。

方法
为探究这一科学问题，本研究对苜蓿疫霉（Phytophthora medicaginis）的基因组进行测序与注释；该菌是引发豆科寄主根腐病并造成显著产量损失的病原菌。我们针对易感鹰嘴豆（Cicer arietinum）寄主的三个定殖阶段的苜蓿疫霉转录组开展分析，并通过共调控分析，在该数量型病害系统中鉴定出可影响BNS阶段时机的潜在小型分泌蛋白（Small Secreted Protein, SSP）效应因子。

结果
苜蓿疫霉的基因组大小约为78 Mb，与同为根腐病致病菌的草莓疫霉（P. fragariae）和悬钩子疫霉（P. rubi）基因组规模相当。尽管基因组规模相近，但其编码的含RxLR（精氨酸-任意氨基酸-亮氨酸-精氨酸）基序的蛋白数量，在已完成全基因组测序的疫霉属物种中位列第二少。目前仅知鹰嘴豆对苜蓿疫霉存在数量抗性，然而本研究发现，侵染过程中大量RxLR效应因子、CRN（Crinkler）效应因子、Nep1样蛋白（Nep1-like protein, NLP）以及碳水化合物活性酶（Carbohydrate Active Enzymes, CAZymes）均受到表达调控。对其中一个编码RxLR效应因子的基因Phytmed_10271进行功能表征后发现，该基因参与调控BNS阶段的时机与根细胞死亡过程。

讨论
本研究结果为阐明仅具备数量遗传抗性的疫霉属病害系统中致病因子的作用，及其对BNS阶段时机的影响提供了重要的研究框架与数据资源。