SignificancePlants employ nucleotide-binding leucine-rich repeat (NLR) immune receptors to recognize pathogen effectors and to activate effector-triggered immunity (ETI). The Toll/IL-1 receptor-NLR (TNL) protein (Roq1) recognizes the effectors XopQ and HopQ1 in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana. Interestingly, we found that the coiled-coil NLR protein N requirement gene 1 (NRG1) is required for activation of ETI by the TNLs Roq1 and Recognition of Peronospora parasitica 1. NRG1 interacts with EDS1 and acts downstream of Roq1 and EDS1 to mediate XopQ/HopQ1-triggered ETI. In addition, Roq1, EDS1, and NRG1 mediate XopQ-triggered transcriptional changes in N. benthamiana and regulate resistance to Xanthomonas and Pseudomonas species that carry the effectors XopQ or HopQ1. This study suggests that NRG1 may be a conserved key component in TNL-mediated signaling pathways.

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Structural biology has the potential to illuminate the evolution of pathogen effectors and their commonalities that cannot be readily detected at the primary sequence level. Recent breakthroughs in protein structure modeling have demonstrated the feasibility to predict the protein folds without depending on homologous templates. These advances enabled a genome-wide computational structural biology approach to help understand proteins based on their predicted folds. In this study, we employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, we predicted the folds of 1,295 proteins (70%). We showed that template-free modeling by TrRosetta captured 514 folds missed by homology modeling, including many known effectors and virulence factors, and that TrRosetta generally produced higher quality models for secreted proteins. Along with sensitive homology search, we employed structure-based clustering, defining not only homologous groups with divergent members but also sequence-unrelated structurally analogous groups. We demonstrate that this approach can reveal new putative members of structurally similar MAX effectors and novel analogous effector families present in M. oryzae and possibly in other phytopathogens. We also investigated the evolution of expanded putative ADP-ribose transferases with predicted structures. We suggest that the loss of catalytic activities of the enzymes might have led them to new evolutionary trajectories to be specialized as protein binders. Collectively, we propose that computational structural genomics approaches can be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of machine learning-based structure prediction. [Formula: see text]Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Elucidating the similarity and diversity of pathogen effectors is critical to understand their evolution across fungal phytopathogens. However, rapid divergence that diminishes sequence similarities between putatively homologous effectors has largely concealed the roots of effector evolution. Here we modelled the structures of 26,653 secreted proteins from 14 agriculturally important fungal phytopathogens, six non-pathogenic fungi and one oomycete with AlphaFold 2. With 18,000 successfully predicted folds, we performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded sequence-unrelated structurally similar (SUSS) effector families and common folds present across the fungal species. Extreme expansion of lineage-specific SUSS effector families was found only in several obligate biotrophs, Blumeria graminis and Puccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified new classes of SUSS effector families that include known virulence factors, such as AvrSr35, AvrSr50 and Tin2. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Putatively sub- and neo-functionalized SUSS effectors could reconverge on regulation, expanding the functional pools of effectors in the pathogen infection cycle. We also found evidence that many effector families could have originated from ancestral folds conserved across fungi. Collectively, our study highlights diverse effector evolution mechanisms and supports divergent evolution as a major force in driving SUSS effector evolution from ancestral proteins.

Background: Nucleotide-binding and leucine-rich repeat immune receptors (NLRs) are an important component of plant immunity that provides resistance against diverse pathogens. NLRs often exist as large gene families, the members of which display diverse multi-domain architectures (MDAs) and evolve through various mechanisms of duplications and selections. Results:We conducted resistance gene enrichment sequencing (RenSeq) with single-molecule real time (SMRT) sequencing of PacBio for 18 accessions in Solanaceae including 15 wild tomatoes. We demonstrate what was previously known as Solanaceae Domain (SD) not only is more diverse in structure and function but also far anciently originated from the most recent common ancestor (MRCA) between Asterids and Amaranthaceae. In tomato, NLRs with the extended N-terminus displayed distinct patterns of evolution based on phylogenetic clades by proliferation, continuous elongation and domain losses. Conclusions:Our study provides high quality gene models of NLRs that can serve as resources for future studies for crop engineering and elucidates greater evolutionary dynamics of the extended NLRs than previously assumed.