RXLR effectors, a class of secreted proteins that are transferred into host cells to manipulate host immunity, have been reported to widely exist in oomycetes, including those from genera Phytophthora, Hyaloperonospora, Albugo, and Saprolegnia. However, in Pythium species, no RXLR effector has yet been characterized, and the origin and evolution of such virulent effectors are still unknown. Here, we developed a modified regular expression method for de novo identification of RXLRs and characterized 359 putative RXLR effectors in nine Pythium species. Phylogenetic analysis revealed that all oomycetous RXLRs formed a single superfamily, suggesting that they might have a common ancestor. RXLR effectors from Pythium and Phytophthora species exhibited similar sequence features, protein structures, and genome locations. In particular, there were significantly more RXLR proteins in the mosquito biological control agent P. guiyangense than in the other eight Pythium species, and P. guiyangense RXLRs might be the result of gene duplication and genome rearrangement events, as indicated by synteny analysis. Expression pattern analysis of RXLR-encoding genes in the plant pathogen P. ultimum detected transcripts of the majority of the predicted RXLR genes, with some RXLR effectors induced in infection stages and one RXLR showing necrosis-inducing activity. Furthermore, all predicted RXLR genes were cloned from two biocontrol agents, P. oligandrum and P. periplocum, and three of the RXLR genes were found to induce a defense response in Nicotiana benthamiana. Taken together, our findings represent the first evidence of RXLR effectors in Pythium species, providing valuable information on their evolutionary patterns and the mechanisms of their interactions with diverse hosts.

As a non-pathogenic oomycete, the biocontrol agent Pythium oligandrum is able to control plant diseases through direct mycoparasite activity and boosting plant immune responses. Several P. oligandrum elicitors have been found to activate plant immunity as microbe-associated molecular patterns (MAMPs). Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are a group of MAMPs widely distributed in eukaryotic and prokaryotic plant pathogens. However, little is known about their distribution and functions in P. oligandrum and its sister species Pythium periplocum. Here, we identified a total of 25 NLPs from P. oligandrum (PyolNLPs) and P. periplocum (PypeNLPs). Meanwhile, we found that PyolNLPs/PypeNLPs genes cluster in two chromosomal segments, and our analysis suggests that they expand by duplication and share a common origin totally different from that of pathogenic oomycetes. Nine PyolNLPs/PypeNLPs induced necrosis in Nicotiana benthamiana by agroinfiltration. Eight partially purified PyolNLPs/PypeNLPs were tested for their potential biocontrol activity. PyolNLP5 and PyolNLP7 showed necrosis-inducing activity in N. benthamiana via direct protein infiltration. At sufficient concentrations, they both significantly reduced disease severity and suppressed the in planta growth of Phytophthora capsici in solanaceous plants including N. benthamiana (tobacco), Solanum lycopersicum (tomato) and Capsicum annuum (pepper). Our assays suggest that the Phytophthora suppression effect of PyolNLP5 and PyolNLP7 is irrelevant to reactive oxygen species (ROS) accumulation. Instead, they induce the expression of antimicrobial plant defensin genes, and the induction depends on their conserved nlp24-like peptide pattern. This work demonstrates the biocontrol role of two P. oligandrum NLPs for solanaceous plants, which uncovers a novel approach of utilizing NLPs to develop bioactive formulae for oomycete pathogen control with no ROS-caused injury to plants.

Elicitins are microbe-associated molecular patterns produced by oomycetes to elicit plant defense. It is still unclear whether elicitins derived from non-pathogenic oomycetes can be used as bioactive molecules for disease control. Here, for the first time we identify and characterize an elicitin named PpEli2 from the soil-borne oomycete Pythium periplocum, which is a non-pathogenic mycoparasite colonizing the root ecosystem of diverse plant species. Perceived by a novel cell surface receptor-like protein, REli, that is conserved in various plants (e.g. tomato, pepper, soybean), PpEli2 can induce hypersensitive response cell death and an immunity response in Nicotiana benthamiana. Meanwhile, PpEli2 enhances the interaction between REli and its co-receptor BAK1. The receptor-dependent immune response triggered by PpEli2 is able to protect various plant species against Phytophthora and fungal infections. Collectively, our work reveals the potential agricultural application of non-pathogenic elicitins and their receptors in conferring broad-spectrum resistance for plant protection.

Being widely existed in oomycetes, the RXLR effector features conserved RXLR-dEER motifs in its N terminal. Every knownPhytophthoraorHyaloperonosporapathogen harbors hundreds of RXLRs. InPythiumspecies, however, none of the RXLR effectors has been characterized yet. Here, we developed a stringent method forde novoidentification of RXLRs and characterized 359 putative RXLR effectors from nine testedPythiumspecies. Phylogenetic analysis revealed a single superfamily formed by all oomycetous RXLRs, suggesting they descent from a common ancestor. RXLR effectors fromPythiumandPhytophthoraspecies exhibited similar sequence features, protein structures and genome locations. In particular, the mosquito biological agentP. guiyangensecontains a significantly larger RXLR repertoire than the other eightPythiumspecies examined, which may result from gene duplication and genome rearrangement events as indicated by synteny analysis. Expression pattern analysis of RXLR-encoding genes in the plant pathogenP. ultimumdetected transcripts from the vast majority of predictedRXLRswith some of them being induced at infection stages. One such RXLRs showed necrosis-inducing activity. Furthermore, all predictedRXLRswere cloned from two biocontrol agentsP. oligandrumandP. periplocum. Three of them were found to encode effectors inducing defense response inNicotiana benthamiana. Taken together, our findings represent the first complete synopsis ofPythiumRXLR effectors, which provides critical clues on their evolutionary patterns as well as the mechanisms of their interactions with diverse hosts.Author summary: Pathogens from thePythiumgenus are widespread across multiple ecological niches. Most of them are soilborne plant pathogens whereas others cause infectious diseases in mammals. SomePythiumspecies can be used as biocontrol agents for plant diseases or mosquito management. Despite that phylogenetically close oomycete pathogens secrete RXLR effectors to enable infection, no RXLR protein was previously characterized in anyPythiumspecies. Here we developed a stringent method to predictPythiumRXLR effectors and compared them with known RXLRs from other species. All oomycetous RXLRs form a huge superfamily, which indicates they may share a common ancestor. Our sequence analysis results suggest that the expansion of RXLR repertoire results from gene duplication and genome recombination events. We further demonstrated that most predictedPythium RXLRscan be transcribed and some of them encode effectors exhibiting pathogenic or defense-inducing activities. This work expands our understanding of RXLR evolution in oomycetes in general, and provides novel insights into the molecular interactions betweenPythiumpathogens and their diverse hosts.