Conserved RxLR Effectors From Oomycetes Hyaloperonospora arabidopsidis and Phytophthora sojae Suppress PAMP- and Effector-Triggered Immunity in Diverse Plants.

Effector proteins are exported to the interior of host cells by diverse plant pathogens. Many oomycete pathogens maintain large families of candidate effector genes, encoding proteins with a secretory leader followed by an RxLR motif. Although most of these genes are very divergent between oomycete species, several genes are conserved between Phytophthora species and Hyaloperonospora arabidopsidis, suggesting that they play important roles in pathogenicity. We describe a pair of conserved effector candidates, HaRxL23 and PsAvh73, from H. arabidopsidis and P. sojae respectively. We show that HaRxL23 is expressed early during infection of Arabidopsis. HaRxL23 triggers an ecotype-specific defense response in Arabidopsis, suggesting that it is recognized by a host surveillance protein. HaRxL23 and PsAvh73 can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Nicotiana benthamiana and effector-triggered immunity (ETI) in soybean. Transgenic Arabidopsis constitutively expressing HaRxL23 or PsAvh73 exhibit suppression of PTI and enhancement of bacterial and oomycete virulence. Together, our experiments demonstrate that these conserved oomycete RxLR effectors suppress PTI and ETI across diverse plant species.

Recent Progress in RXLR Effector Research.

Some of the most devastating oomycete pathogens deploy effector proteins, with the signature amino acid motif RXLR, that enter plant cells to promote virulence. Research on the function and evolution of RXLR effectors has been very active over the decade that has transpired since their discovery. Comparative genomics indicate that RXLR genes play a major role in virulence for Phytophthora and downy mildew species. Importantly, gene-for-gene resistance against these oomycete lineages is based on recognition of RXLR proteins. Comparative genomics have revealed several mechanisms through which this resistance can be broken, most notably involving epigenetic control of RXLR gene expression. Structural studies have revealed a core fold that is present in the majority of RXLR proteins, providing a foundation for detailed mechanistic understanding of virulence and avirulence functions. Finally, functional studies have demonstrated that suppression of host immunity is a major function for RXLR proteins. Host protein targets are being identified in a variety of plant cell compartments. Some targets comprise hubs that are also manipulated by bacteria and fungi, thereby revealing key points of vulnerability in the plant immune network.

Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants.

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR-like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non-pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's host.

Application of alignment-free bioinformatics methods to identify an oomycete protein with structural and functional similarity to the bacterial AvrE effector protein.

Diverse plant pathogens export effector proteins to reprogram host cells. One of the most challenging goals in the molecular plant-microbe field is to functionally characterize the complex repertoires of effectors secreted by these pathogens. For bacterial pathogens, the predominant class of effectors is delivered to host cells by Type III secretion. For oomycetes, the predominant class of effectors is defined by a signal peptide that mediates secretion from the oomycete and a conserved RxLR motif. Downy mildew pathogens and Phytophthora species maintain hundreds of candidate RxLR effector genes in their genomes. Although no primary sequence similarity is evident between bacterial Type III effectors (T3Es) and oomycete RXLR effectors, some bacterial and oomycete effectors have convergently evolved to target the same host proteins. Such effectors might have evolved domains that are functionally similar but sequence-unrelated. We reasoned that alignment-free bioinformatics approaches could be useful to identify structural similarities between bacterial and oomycete effectors. To test this approach, we used partial least squares regression, alignment-free bioinformatics methods to identify effector proteins from the genome of the oomycete Hyaloperonospora arabidopsidis that are similar to the well-studied AvrE1 effector from Pseudomonas syringae. This approach identified five RxLR proteins with putative structural similarity to AvrE1. We focused on one, HaRxL23, because it is an experimentally validated effector and it is conserved between distantly related oomycetes. Several experiments indicate that HaRxL23 is functionally similar to AvrE1, including the ability to partially rescue an AvrE1 loss-of-function mutant. This study provides an example of how an alignment-free bioinformatics approach can identify functionally similar effector proteins in the absence of primary sequence similarity. This approach could be useful to identify effectors that have convergently evolved regardless of whether the shared host target is known.