Identification And Functional Analysis Of The Plant Interacting Factors Of Phytophthora Capsici Effector PcAvh1

The oomycete Phytophthora capsici Leonian is an important plant pathogen that often attacks a variety of crops including many of solanaceous, cucurbit and legume species. During the interaction between P. capsici and host plants, P. capsici secretes a large number of effectors into the host cells to suppress the immune defense reaction of the host plants and to promote the infection and colonization by the pathogen. In order to resist the invasion of the pathogen, the plants activate their immune response. Therefore, understanding the molecular mechanisms underlying the Phytophthora-plant interactions is important for elucidating the pathogenesis of Phytophthora and designing the control strategy of diseases.RXLR proteins are an important class of oomycetes cytoplasmic effectors, which could trigger or suppress the defense response of host plants when they were delivered into the host cells. By doing this, RXLR effectors help the pathogens to infect and colonize plants, leading to the diseases. In previous studies, we found that one P. capsici RXLR effector PcAvhl could trigger plant cell death (PCD) response in model plant Nicotiana benthamiana. Silencing of PcAvhl gene significantly impaired the virulence of P. capsici on Capsicum annuum and N. benthamiana. The above results showed that PcAvh1 is an important virulence factor in P. capsici. However, it is unclear how this effector is involved in the pathogenesis of P. capsici. The study here provides important experimental evidence to understand the pathogenesis mechanism of P. capsici by identification and functional analysis of the interactors of PcAvhl in plants.The study first analyzed whether PcAvhl could trigger PCD on Nicotiana tabacum, Capsicum annuum and Solanum lycopersicum besides N. benthamiana by using the Agrobacterium-mediated transient expression. We found that PcAvh1 can trigger PCD on three tomato varieties (L402, Zhongza 9 and Maofen 802) and cause serious yellowing phenomenon on two pepper varieties (ox horn pepper and sweet pepper) while it did not cause obvious symptomatic phenotypes on tobacco. The present study then screened the N. benthamiana infection cDNA library by P. capsici by yeast two-hybrid assay using PcAvhl as the bait. Two target proteins (serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A, PP2A-1 and one isoform, PP2A-2) interacting with PcAvh1 were obtained. The bimolecular fluorescence complementation assy verified the interaction between PP2As and PcAvhl in vivo.qRT-PCR was used to examine the expression levels of PP2As during the infection stages of N. benthamiana by P. capsici (1.5,3,6,12,24,36 and 72 h post-inoculation) and different growth stages of N. benthamiana. It was found that PP2As were differentially expressed when N. benthamiana was infected by P. capsici. In addition to this, PP2As had the highest expression in the flowers of N. benthamiana over other four tissues (root, stem, leaf and seed).Virus-induced gene silencing was used to further analyze the function of interacting proteins. It was found that the growth of N. benthamiana was normal when single PP2A gene (PP2A-1 or PP2A-2) was silenced. However, the N. benthamiana plants were severely dwarf when both PP2A genes were silenced. All PP2A-silenced plants demonstrated decreased resistance to the infection of P. capsici. However, it is found that PP2A-silencing does not affect PCD triggered by PcAvhl in N. benthamiana. These results showed that PP2As are probably an important class of protein phosphatases, which play important roles in the defending plants against pathogens.In this study, two interacting proteins of PcAvhl were obtained by yeast two-hybrid. Their differential expression was observed during the infection stages of N. benthamiana by P. capsici and among different tissues of N. benthamiana using qRT-PCR assay. Furthermore, by virus-induced gene silencing technology, it is revealed that PP2As play an important role in the disease resistance of N. benthamiana against the pathogen infection. The study provides important experimental data for elucidating the interaction mechanism between P. capsici and plants, and is valuable to the development of the disease control strategy.