The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

Summary The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N‐terminal motif within Avh241 was important for membrane localization and cell death‐inducing activity. Two mitogen‐activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen’s full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization.

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

Mangrove roots harbor a repertoire of microbial taxa that contribute to important ecological functions in mangrove ecosystems. However, the diversity, function, and assembly of mangrove root-associated microbial communities along a continuous fine-scale niche remain elusive. Here, we applied amplicon and metagenome sequencing to investigate the bacterial and fungal communities among four compartments (nonrhizosphere, rhizosphere, episphere, and endosphere) of mangrove roots. We found different distribution patterns for both bacterial and fungal communities in all four root compartments, which could be largely due to niche differentiation along the root compartments and exudation effects of mangrove roots. The functional pattern for bacterial and fungal communities was also divergent within the compartments. The endosphere harbored more genes involved in carbohydrate metabolism, lipid transport, and methane production, and fewer genes were found to be involved in sulfur reduction compared to other compartments. The dynamics of root-associated microbial communities revealed that 56–74% of endosphere bacterial taxa were derived from nonrhizosphere, whereas no fungal OTUs of nonrhizosphere were detected in the endosphere. This indicates that roots may play a more strictly selective role in the assembly of the fungal community compared to the endosphere bacterial community, which is consistent with the projections established in an amplification-selection model. This study reveals the divergence in the diversity and function of root-associated microbial communities along a continuous fine-scale niche, thereby highlighting a strictly selective role of soil-root interfaces in shaping the fungal community structure in the mangrove root systems.