A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.

Rapid dissemination of host metabolism-manipulating genes via integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

Molecular basis for the interference of the Arabidopsis WRKY54-mediated immune response by two sequence-unrelated bacterial effectors.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

Investigation of natural RIN4 variants reveals a motif crucial for function and provides an opportunity to broaden NLR regulation specificity.

Multiple bacterial effectors target RPM1-INTERACTING PROTEIN4 (RIN4), the biochemical modifications of which are recognized by several plant nucleotide-binding and leucine-rich repeat immune receptor (NLR) proteins. Recently, a comparative study of Arabidopsis and apple (Malus domestica) RIN4s revealed that the RIN4 specificity motif (RSM) is critical for NLR regulation. Here, we investigated the extent to which the RSM contributes to the functions of natural RIN4 variants. Functional analysis of 33 natural RIN4 variants from 28 plant species showed that the RSM is generally required yet sometimes dispensable for the RIN4-mediated suppression of NLR auto-activity or effector-triggered NLR activation. Association analysis of the sequences and fire blight resistance gene originating from Malus × robusta 5 (FB_MR5) activation functions of the natural RIN4 variants revealed H167 to be an indispensable residue for RIN4 function in the regulation of NLRs. None of the tested natural RIN4 variants could suppress RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA1 (RPM1) auto-activity and activate FB_MR5. To engineer RIN4 to carry broader NLR compatibility, we generated chimeric RIN4 proteins, several of which could regulate RPM1, RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2), and FB_MR5. We propose that the intrinsically disordered nature of RIN4 provides a flexible platform to broaden pathogen recognition specificity by establishing compatibility with otherwise incompatible NLRs.

Engineering homoeologs provide a fine scale for quantitative traits in polyploid.

Numerous staple crops exhibit polyploidy and are difficult to genetically modify. However, recent advances in genome sequencing and editing have enabled polyploid genome engineering. The hexaploid black nightshade species Solanum nigrum has immense potential as a beneficial food supplement. We assembled its genome at the scaffold level. After functional annotations, we identified homoeologous gene sets, with similar sequence and expression profiles, based on comparative analyses of orthologous genes with close diploid relatives Solanum americanum and S. lycopersicum. Using CRISPR-Cas9-mediated mutagenesis, we generated various mutation combinations in homoeologous genes. Multiple mutants showed quantitative phenotypic changes based on the genotype, resulting in a broad-spectrum effect on the quantitative traits of hexaploid S. nigrum. Furthermore, we successfully improved the fruit productivity of Boranong, an orphan cultivar of S. nigrum suggesting that engineering homoeologous genes could be useful for agricultural improvement of polyploid crops.

Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities.

Some nucleotide-binding and leucine-rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence-unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4-targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library. We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB. We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1-dependent recognition of AvrBsT and HopZ5. The recognition of sequence-unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1-mediated immunity could reveal unique mechanisms of expanded effector recognition.

Ralstonia solanacearum Type III Effector RipJ Triggers Bacterial Wilt Resistance in Solanum pimpinellifolium.

Ralstonia solanacearum causes bacterial wilt disease in solanaceous crops. Identification of avirulence type III-secreted effectors recognized by specific disease resistance proteins in host plant species is an important step toward developing durable resistance in crops. In the present study, we show that R. solanacearum effector RipJ functions as an avirulence determinant in Solanum pimpinellifolium LA2093. In all, 10 candidate avirulence effectors were shortlisted based on the effector repertoire comparison between avirulent Pe_9 and virulent Pe_1 strains. Infection assays with transgenic strain Pe_1 individually carrying a candidate avirulence effector from Pe_9 revealed that only RipJ elicits strong bacterial wilt resistance in S. pimpinellifolium LA2093. Furthermore, we identified that several RipJ natural variants do not induce bacterial wilt resistance in S. pimpinellifolium LA2093. RipJ belongs to the YopJ family of acetyltransferases. Our sequence analysis indicated the presence of partially conserved putative catalytic residues. Interestingly, the conserved amino acid residues in the acetyltransferase catalytic triad are not required for effector-triggered immunity. In addition, we show that RipJ does not autoacetylate its lysine residues. Our study reports the identification of the first R. solanacearum avirulence protein that triggers bacterial wilt resistance in tomato. We expect that our discovery of RipJ as an avirulence protein will accelerate the development of bacterial wilt-resistant tomato varieties in the future.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Phylogenetic analysis of ABCG subfamily proteins in plants: functional clustering and coevolution with ABCGs of pathogens.

ABCG subfamily proteins are highly enriched in terrestrial plants. Many of these proteins secrete secondary metabolites that repel or inhibit pathogens. To establish why the ABCG subfamily proteins proliferated extensively during evolution, we constructed phylogenetic trees from a broad range of eukaryotic organisms. ABCG proteins were massively duplicated in land plants and in oomycetes, a group of agronomically important plant pathogens, which prompted us to hypothesize that plant and pathogen ABCGs coevolved. Supporting this hypothesis, full-size ABCGs in host plants (Arabidopsis thaliana and Glycine max) and their pathogens (Hyaloperonospora arabidopsidis and Phytophthora sojae, respectively) had similar divergence times and patterns. Furthermore, generalist pathogens with broad ranges of host plants have diversified more ABCGs than their specialist counterparts. The hypothesis was further tested using an example pair of ABCGs that first diverged during multiplication in a host plant and its pathogen: AtABCG31 of A. thaliana and HpaP802307 of H. arabidopsidis. AtABCG31 expression was activated following infection with H. arabidopsidis, and disrupting AtABCG31 led to increased susceptibility to H. arabidopsidis. Together, our results suggest that ABCG genes in plants and their oomycete pathogens coevolved in an arms race, to extrude secondary metabolites involved in the plant's defense response against pathogens.

Improved Genome Sequence and Gene Annotation Resource for the Potato Late Blight Pathogen Phytophthora infestans.

Phytophthora infestans is a devastating pathogen causing potato late blight (Solanum tuberosum). Here we report the sequencing, assembly and genome annotation for two Phytophthora infestans isolates sampled in Republic of Korea. Genome sequencing was carried out using long read (Oxford Nanopore) and short read (Illumina Nextseq) sequencing technologies that significantly improved the contiguity and quality of P. infestans genome assembly. Our resources would help researchers better understand the molecular mechanisms by which P. infestans causes late blight disease in the future.

What are the Top 10 Unanswered Questions in Molecular Plant-Microbe Interactions?

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A host target of a bacterial cysteine protease virulence effector plays a key role in convergent evolution of plant innate immune system receptors.

Some virulence effectors secreted from pathogens target host proteins and induce biochemical modifications that are monitored by nucleotide-binding and leucine-rich repeat (NLR) immune receptors. Arabidopsis RIN4 protein (AtRIN4: RPM1-interacting protein 4) homologs are present in diverse plant species and targeted by several bacterial type III effector proteins including the cysteine protease AvrRpt2. RIN4 is 'guarded' by several independently evolved NLRs from various plant species, including Arabidopsis RPS2. Recently, it was shown that the MR5 NLR from a wild apple relative can recognize the AvrRpt2 effector from Erwinia amylovora, but the details of this recognition remained unclear. The present contribution reports the mechanism of AvrRpt2 recognition by independently evolved NLRs, MR5 from apple and RPS2, both of which require proteolytically processed RIN4 for activation. It shows that the C-terminal cleaved product of apple RIN4 (MdRIN4) but not AtRIN4 is necessary and sufficient for MR5 activation. Additionally, two polymorphic residues in AtRIN4 and MdRIN4 are identified that are crucial in the regulation of and physical association with NLRs. It is proposed that polymorphisms in RIN4 from distantly related plant species allow it to remain an effector target while maintaining compatibility with multiple NLRs.

TGFam-Finder: a novel solution for target-gene family annotation in plants.

Whole-genome annotation error that omits essential protein-coding genes hinders further research. We developed Target Gene Family Finder (TGFam-Finder), an alternative tool for the structural annotation of protein-coding genes containing target domain(s) of interest in plant genomes. TGFam-Finder took considerably reduced annotation run-time and improved accuracy compared to conventional annotation tools. Large-scale re-annotation of 50 plant genomes identified an average of 150, 166 and 86 additional far-red-impaired response 1, nucleotide-binding and leucine-rich-repeat, and cytochrome P450 genes, respectively, that were missed in previous annotations. We detected significantly higher number of translated genes in the new annotations using mass spectrometry data from seven plant species compared to previous annotations. TGFam-Finder along with the new gene models can provide an optimized platform for comprehensive functional, comparative, and evolutionary studies in plants.

Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin.

Plant pathogens cause huge yield losses. Plant defense often depends on toxic secondary metabolites that inhibit pathogen growth. Because most secondary metabolites are also toxic to the plant, specific transporters are needed to deliver them to the pathogens. To identify the transporters that function in plant defense, we screened Arabidopsis thaliana mutants of full-size ABCG transporters for hypersensitivity to sclareol, an antifungal compound. We found that atabcg34 mutants were hypersensitive to sclareol and to the necrotrophic fungi Alternaria brassicicola and Botrytis cinereaAtABCG34 expression was induced by Abrassicicola inoculation as well as by methyl-jasmonate, a defense-related phytohormone, and AtABCG34 was polarly localized at the external face of the plasma membrane of epidermal cells of leaves and roots. atabcg34 mutants secreted less camalexin, a major phytoalexin in Athaliana, whereas plants overexpressing AtABCG34 secreted more camalexin to the leaf surface and were more resistant to the pathogen. When treated with exogenous camalexin, atabcg34 mutants exhibited hypersensitivity, whereas BY2 cells expressing AtABCG34 exhibited improved resistance. Analyses of natural Arabidopsis accessions revealed that AtABCG34 contributes to the disease resistance in naturally occurring genetic variants, albeit to a small extent. Together, our data suggest that AtABCG34 mediates camalexin secretion to the leaf surface and thereby prevents Abrassicicola infection.

Multiple functional self-association interfaces in plant TIR domains.

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

High Contiguity Whole Genome Sequence and Gene Annotation Resource for Two Venturia nashicola Isolates.

Venturia nashicola is a fungal pathogen that causes Asian pear scab disease. This pathogen is of particular importance in Northeast Asian countries, where Asian pears are grown industrially. Scab disease in Asian pear is currently controlled by fungicide spraying and this situation calls for developing scab resistant cultivars. High-quality genome data are therefore required for in-depth comparative genome analysis of different isolates of V. nashicola and V. pyrina, a closely related species, which only infects European pear plants. Here, we report the high-contiguity whole genome assembly of two V. nashicola isolates, which is expected to enable genome comparisons for identification of the genes involved in host range determination of V. nashicola.

Autoimmunity and effector recognition in Arabidopsis thaliana can be uncoupled by mutations in the RRS1-R immune receptor.

Plant nucleotide-binding leucine-rich repeat (NLR) disease resistance proteins recognize specific pathogen effectors and activate a cellular defense program. In Arabidopsis thaliana (Arabidopsis), Resistance to Ralstonia solanacearum 1 (RRS1-R) and Resistance to Pseudomonas syringae 4 (RPS4) function together to recognize the unrelated bacterial effectors PopP2 and AvrRps4. In the plant cell nucleus, the RRS1-R/RPS4 complex binds to and signals the presence of AvrRps4 or PopP2. The exact mechanism underlying NLR signaling and immunity activation remains to be elucidated. Using genetic and biochemical approaches, we characterized the intragenic suppressors of sensitive to low humidity 1 (slh1), a temperature-sensitive autoimmune allele of RRS1-R. Our analyses identified five amino acid residues that contribute to RRS1-RSLH1 autoactivity. We investigated the role of these residues in the RRS1-R allele by genetic complementation, and found that C15 in the Toll/interleukin-1 receptor (TIR) domain and L816 in the LRR domain were also important for effector recognition. Further characterization of the intragenic suppressive mutations located in the RRS1-R TIR domain revealed differing requirements for RRS1-R/RPS4-dependent autoimmunity and effector-triggered immunity. Our results provide novel information about the mechanisms which, in turn, hold an NLR protein complex inactive and allow adequate activation in the presence of pathogens.

Arabidopsis thaliana SOBER1 (SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1) suppresses plant immunity triggered by multiple bacterial acetyltransferase effectors.

Plants evolved disease resistance (R) proteins that recognize corresponding pathogen effectors and activate effector-triggered immunity (ETI). However, it is largely unknown why, in some cases, a suppressor of ETI exists in plants. Arabidopsis SOBER1 (Suppressor of AvrBsT-elicited Resistance 1) was identified previously as a suppressor of Xanthomonas acetyltransferase effector AvrBsT-triggered immunity. Nevertheless, the extent to which SOBER1 suppresses ETI is unclear. Here, we identified SOBER1 as a suppressor of Pseudomonas acetyltransferase effector HopZ5-triggered immunity in Arabidopsis using recombinant inbred lines. Further analysis showed that SOBER1 suppresses immunity triggered by multiple bacterial acetyltransferases. Interestingly, SOBER1 interferes with the immunity signalling activated by some but not all tested acetyltransferase effectors, indicating that SOBER1 might target components that are shared between several ETI pathways.

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific "avirulent" pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.

Distinct regions of the Pseudomonas syringae coiled-coil effector AvrRps4 are required for activation of immunity.

Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding-leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4-dependent immunity in resistant accessions of Arabidopsis. To better understand the molecular basis of AvrRps4-triggered immunity, we determined the crystal structure of processed AvrRps4 (AvrRps4(C), residues 134-221), revealing that it forms an antiparallel α-helical coiled coil. Structure-informed mutagenesis reveals an electronegative surface patch in AvrRps4(C) required for recognition by RPS4; mutations in this region can also uncouple triggering of the hypersensitive response from disease resistance. This uncoupling may result from a lower level of defense activation, sufficient for avirulence but not for triggering a hypersensitive response. Natural variation in AvrRps4 reveals distinct recognition specificities that involve a surface-exposed residue. Recently, a direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 has been implicated in activation of immunity. However, we were unable to detect direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 after coexpression in Nicotiana benthamiana or in yeast cells. How intracellular plant immune receptors activate defense upon effector perception remains an unsolved problem. The structure of AvrRps4(C), and identification of functionally important residues for its activation of plant immunity, advances our understanding of these processes in a well-defined model pathosystem.

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants.