Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed Microbe-Associated Molecular Patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multi-copy MAMPs on immune induction is unknown. Here we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple Cold Shock Proteins and 46% carry a non-immunogenic form. We uncovered a new mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic Cold Shock Protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Variation in microbial feature perception in the Rutaceae family with immune receptor conservation in citrus.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in 2 subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22), and chitin, including 1 feature from Candidatus Liberibacter species (csp22CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized 2 genetically linked FLS2 homologs from "Frost Lisbon" lemon (Citrus ×limon, responsive) and "Washington navel" orange (Citrus ×aurantium, nonresponsive). Surprisingly, FLS2 homologs from responsive and nonresponsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. "Washington navel" orange weakly responded to chitin, whereas "Tango" mandarin (C. ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the 2 genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Overlapping Local and Systemic Defense Induced by an Oomycete Fatty Acid MAMP and Brown Seaweed Extract in Tomato.

Eicosapolyenoic fatty acids are integral components of oomycete pathogens that can act as microbe-associated molecular patterns to induce disease resistance in plants. Defense-inducing eicosapolyenoic fatty acids include arachidonic acid (AA) and eicosapentaenoic acid and are strong elicitors in solanaceous plants, with bioactivity in other plant families. Similarly, extracts of a brown seaweed, Ascophyllum nodosum, used in sustainable agriculture as a biostimulant of plant growth, may also induce disease resistance. A. nodosum, similar to other macroalgae, is rich in eicosapolyenoic fatty acids, which comprise as much as 25% of total fatty acid composition. We investigated the response of roots and leaves from AA or a commercial A. nodosum extract (ANE) on root-treated tomatoes via RNA sequencing, phytohormone profiling, and disease assays. AA and ANE significantly altered transcriptional profiles relative to control plants, inducing numerous defense-related genes with both substantial overlap and differences in gene expression patterns. Root treatment with AA and, to a lesser extent, ANE also altered both salicylic acid and jasmonic acid levels while inducing local and systemic resistance to oomycete and bacterial pathogen challenge. Thus, our study highlights overlap in both local and systemic defense induced by AA and ANE, with potential for inducing broad-spectrum resistance against pathogens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid.

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.[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.

Stress-induced reactive oxygen species compartmentalization, perception and signalling.

Reactive oxygen species (ROS) are essential for life and are involved in the regulation of almost all biological processes. ROS production is critical for plant development, response to abiotic stresses and immune responses. Here, we focus on recent discoveries in ROS biology emphasizing abiotic and biotic stress responses. Recent advancements have resulted in the identification of one of the first sensors for extracellular ROS and highlighted waves of ROS production during stress signalling in Arabidopsis. Enzymes that produce ROS, including NADPH oxidases, exhibit precise regulation through diverse post-translational modifications. Discoveries highlight the importance of both amino- and carboxy-terminal regulation of NADPH oxidases through protein phosphorylation and cysteine oxidation. Here, we discuss advancements in ROS compartmentalization, systemic ROS waves, ROS sensing and post-translational modification of ROS-producing enzymes and identify areas where foundational gaps remain.

Phosphorylation of the Pseudomonas Effector AvrPtoB by Arabidopsis SnRK2.8 Is Required for Bacterial Virulence.

A critical component controlling bacterial virulence is the delivery of pathogen effectors into plant cells during infection. Effectors alter host metabolism and immunity for the benefit of pathogens. Multiple effectors are phosphorylated by host kinases, and this posttranslational modification is important for their activity. We sought to identify host kinases involved in effector phosphorylation. Multiple phosphorylated effector residues matched the proposed consensus motif for the plant calcium-dependent protein kinase (CDPK) and Snf1-related kinase (SnRK) superfamily. The conserved Pseudomonas effector AvrPtoB acts as an E3 ubiquitin ligase and promotes bacterial virulence. In this study, we identified a member of the Arabidopsis SnRK family, SnRK2.8, which interacts with AvrPtoB in yeast and in planta. We showed that SnRK2.8 was required for AvrPtoB virulence functions, including facilitating bacterial colonization, suppression of callose deposition, and targeting the plant defense regulator NPR1 and analyses receptor FLS2. Mass spectrometry analysis revealed that AvrPtoB phosphorylation occurs at multiple serine residues in planta, with S258 phosphorylation significantly reduced in the snrk2.8 knockout. AvrPtoB phospho-null mutants exhibited compromised virulence functions and were unable to suppress NPR1 accumulation, FLS2 accumulation, or inhibit FLS2-BAK1 complex formation upon flagellin perception. Taken together, these data identify a conserved plant kinase utilized by a pathogen effector to promote disease.

Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression.

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

Phytopathogenic Rhodococcus Have Diverse Plasmids With Few Conserved Virulence Functions.

Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kb-sized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.

Interspecific Gene Exchange Introduces High Genetic Variability in Crop Pathogen.

Genome analyses have revealed a profound role of hybridization and introgression in the evolution of many eukaryote lineages, including fungi. The impact of recurrent introgression on fungal evolution however remains elusive. Here, we analyzed signatures of introgression along the genome of the fungal wheat pathogen Zymoseptoria tritici. We applied a comparative population genomics approach, including genome data from five Zymoseptoria species, to characterize the distribution and composition of introgressed regions representing segments with an exceptional haplotype pattern. These regions are found throughout the genome, comprising 5% of the total genome and overlapping with > 1,000 predicted genes. We performed window-based phylogenetic analyses along the genome to distinguish regions which have a monophyletic or nonmonophyletic origin with Z. tritici sequences. A majority of nonmonophyletic windows overlap with the highly variable regions suggesting that these originate from introgression. We verified that incongruent gene genealogies do not result from incomplete lineage sorting by comparing the observed and expected length distribution of haplotype blocks resulting from incomplete lineage sorting. Although protein-coding genes are not enriched in these regions, we identify 18 that encode putative virulence determinants. Moreover, we find an enrichment of transposable elements in these regions implying that hybridization may contribute to the horizontal spread of transposable elements. We detected a similar pattern in the closely related species Zymoseptoria ardabiliae, suggesting that hybridization is widespread among these closely related grass pathogens. Overall, our results demonstrate a significant impact of recurrent hybridization on overall genome evolution of this important wheat pathogen.

The Evolution, Ecology, and Mechanisms of Infection by Gram-Positive, Plant-Associated Bacteria.

Gram-positive bacteria are prominent members of plant-associated microbial communities. Although many are hypothesized to be beneficial, some are causative agents of economically important diseases of crop plants. Because the features of Gram-positive bacteria are fundamentally different relative to those of Gram-negative bacteria, the evolution and ecology as well as the mechanisms used to colonize and infect plants also differ. Here, we discuss recent advances in our understanding of Gram-positive, plant-associated bacteria and provide a framework for future research directions on these important plant symbionts.

Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management.

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.