The Ralstonia pseudosolanacearum Type III Effector RipL Delays Flowering and Promotes Susceptibility to Pseudomonas syringae in Arabidopsis thaliana.

The plant defense responses to microbial infection are tightly regulated and integrated with the developmental program for optimal resources allocation. Notably, the defense- associated hormone salicylic acid (SA) acts as a promoter of flowering while several plant pathogens actively target the flowering signaling pathway to promote their virulence or dissemination. Ralstonia pseudosolanacearum inject tens of effectors in the host cells that collectively promote bacterial proliferation in plant tissues. Here, we characterized the function of the broadly conserved R. pseudosolanacearum effector RipL, through heterologous expression in Arabidopsis thaliana . RipL-expressing transgenic lines presented a delayed flowering, which correlated with a low expression of flowering regulator genes. Delayed flowering was also observed in Nicotiana benthamiana plants transiently expressing RipL. In parallel, RipL promoted plant susceptibility to virulent strains of Pseudomonas syringae in the effector-expressing lines or when delivered by the type III secretion system. Unexpectedly, SA accumulation and SA-dependent immune signaling were not significantly affected by RipL expression. Rather, the RNA-seq analysis of infected RipL-expressing lines revealed that the overall amplitude of the transcriptional response was dampened, suggesting that RipL could promote plant susceptibility in an SA-independent manner. Further elucidation of the molecular mechanisms underpinning RipL effect on flowering and immunity may reveal novel effector functions in host cells.

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.

Diversified host target families mediate convergently evolved effector recognition across plant species.

Recognition of pathogen effectors is a crucial step for triggering plant immunity. Resistance (R) genes often encode for nucleotide-binding leucine-rich repeat receptors (NLRs), and NLRs detect effectors from pathogens to trigger effector-triggered immunity (ETI). NLR recognition of effectors is observed in diverse forms where NLRs directly interact with effectors or indirectly detect effectors by monitoring host guardees/decoys (HGDs). HGDs undergo different biochemical modifications by diverse effectors and expand the effector recognition spectrum of NLRs, contributing robustness to plant immunity. Interestingly, in many cases of the indirect recognition of effectors, HGD families targeted by effectors are conserved across the plant species while NLRs are not. Notably, a family of diversified HGDs can activate multiple non-orthologous NLRs across plant species. Further investigation on HGDs would reveal the mechanistic basis of how the diversification of HGDs confers novel effector recognition by NLRs.

A plasma membrane nucleotide-binding leucine-rich repeat receptor mediates the recognition of the Ralstonia pseudosolanacearum effector RipY in Nicotiana benthamiana.

Bacterial wilt disease caused by several Ralstonia species is one of the most destructive diseases in Solanaceae crops. Only a few functional resistance genes against bacterial wilt have been cloned to date. Here, we show that the broadly conserved type III secreted effector RipY is recognized by the Nicotiana benthamiana immune system, leading to cell death induction, induction of defense-related gene expression, and restriction of bacterial pathogen growth. Using a multiplexed virus-induced gene-silencing-based N. benthamiana nucleotide-binding and leucine-rich repeat receptor (NbNLR) library, we identified a coiled-coil (CC) nucleotide-binding and leucine-rich repeat receptor (CNL) required for recognition of RipY, which we named RESISTANCE TO RALSTONIA SOLANACEARUM RIPY (RRS-Y). Genetic complementation assays in RRS-Y-silenced plants and stable rrs-y knockout mutants demonstrated that RRS-Y is sufficient to activate RipY-induced cell death and RipY-induced immunity to Ralstonia pseudosolanacearum. RRS-Y function is dependent on the phosphate-binding loop motif of the nucleotide-binding domain but independent of the characterized signaling components ENHANCED DISEASE SUSCEPTIBILITY 1, ACTIVATED DISEASE RESISTANCE 1, and N REQUIREMENT GENE 1 and the NLR helpers NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2, -3, and -4 in N. benthamiana. We further show that RRS-Y localization at the plasma membrane is mediated by two cysteine residues in the CC domain and is required for RipY recognition. RRS-Y also broadly recognizes RipY homologs across Ralstonia species. Lastly, we show that the C-terminal region of RipY is indispensable for RRS-Y activation. Together, our findings provide an additional effector/receptor pair system to deepen our understanding of CNL activation in plants.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1.

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus-induced gene silencing of the nucleotide-binding and leucine-rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1-induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb-ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1-dependent recognition. Furthermore, NbPtr1-dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae.

An Alternative Method to Evaluate Resistance to Pear Scab (Venturia nashicola).

Two pear cultivars with different degrees of resistance to Venturia nashicola were evaluated on the basis of a disease severity rating for pear scab resistance under controlled environmental condition. Two inoculation techniques were tested: the procedure for inoculation by dropping conidia suspension of V. nashicola; the procedure by deposition of agar plug on the abaxial surface of pear leaves. All tested cultivars resulted in blight symptoms on the inoculated leaves and became spread to uninoculated region or other leaves. Although both methods provide satisfactory infection of V. nashicola on pear leaves, the mycelial plug method of inoculation was more reliable than the spray inoculation method for the evaluation of pear scab disease resistance. The incubation period of V. nashicola in the resistant pear cultivar, Greensis was longer than that in the susceptible cultivar, Hwasan.

Manipulation of the Host Endomembrane System by Bacterial Effectors.

The endomembrane system, extending from the nuclear envelope to the plasma membrane, is critical to the plant response to pathogen infection. Synthesis and transport of immunity-related proteins and antimicrobial compounds to and from the plasma membrane are supported by conventional and unconventional processes of secretion and internalization of vesicles, guided by the cytoskeleton networks. Although plant bacterial pathogens reside mostly in the apoplast, major structural and functional modifications of the endomembrane system in the host cell occur during bacterial infection. Here, we review the dynamics of these cellular compartments, briefly, for their essential contributions to the plant defense responses and, in parallel, for their emerging roles in bacterial pathogenicity. We further focus on Pseudomonas syringae, Xanthomonas spp., and Ralstonia solanacearum type III secreted effectors that one or both localize to and associate with components of the host endomembrane system or the cytoskeleton network to highlight the diversity of virulence strategies deployed by bacterial pathogens beyond the inhibition of the secretory pathway. [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.

The Phytophthora capsici RxLR effector CRISIS2 triggers cell death via suppressing plasma membrane H+-ATPase in the host plant.

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.

The Danger-Associated Peptide PEP1 Directs Cellular Reprogramming in the Arabidopsis Root Vascular System.

When perceiving microbe-associated molecular patterns (MAMPs) or plant-derived damage-associated molecular patterns (DAMPs), plants alter their root growth and development by displaying a reduction in the root length and the formation of root hairs and lateral roots. The exogenous application of a MAMP peptide, flg22, was shown to affect root growth by suppressing meristem activity. In addition to MAMPs, the DAMP peptide PEP1 suppresses root growth while also promoting root hair formation. However, the question of whether and how these elicitor peptides affect the development of the vascular system in the root has not been explored. The cellular receptors of PEP1, PEPR1 and PEPR2 are highly expressed in the root vascular system, while the receptors of flg22 (FLS2) and elf18 (EFR) are not. Consistent with the expression patterns of PEP1 receptors, we found that exogenously applied PEP1 has a strong impact on the division of stele cells, leading to a reduction of these cells. We also observed the alteration in the number and organization of cells that differentiate into xylem vessels. These PEP1-mediated developmental changes appear to be linked to the blockage of symplastic connections triggered by PEP1. PEP1 dramatically disrupts the symplastic movement of free green fluorescence protein (GFP) from phloem sieve elements to neighboring cells in the root meristem, leading to the deposition of a high level of callose between cells. Taken together, our first survey of PEP1-mediated vascular tissue development provides new insights into the PEP1 function as a regulator of cellular reprogramming in the Arabidopsis root vascular system.

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.

Identification of RipAZ1 as an avirulence determinant of Ralstonia solanacearum in Solanum americanum.

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease.

Perception of unrelated microbe-associated molecular patterns triggers conserved yet variable physiological and transcriptional changes in Brassica rapa ssp. pekinensis.

Pattern-triggered immunity (PTI) includes the different transcriptional and physiological responses that enable plants to ward off microbial invasion. Surface-localized pattern-recognition receptors (PRRs) recognize conserved microbe-associated molecular patterns (MAMPs) and initiate a branched signaling cascade that culminate in an effective restriction of pathogen growth. In the model species Arabidopsis thaliana, early PTI events triggered by different PRRs are broadly conserved although their nature or intensity is dependent on the origin and features of the detected MAMP. In order to provide a functional basis for disease resistance in leafy vegetable crops, we surveyed the conservation of PTI events in Brassica rapa ssp. pekinensis. We identified the PRR homologs present in B. rapa genome and found that only one of the two copies of the bacterial Elongation factor-Tu receptor (EFR) might function. We also characterized the extent and unexpected specificity of the transcriptional changes occurring when B. rapa seedlings are treated with two unrelated MAMPs, the bacterial flagellin flg22 peptide and the fungal cell wall component chitin. Finally, using a MAMP-induced protection assay, we could show that bacterial and fungal MAMPs elicit a robust immunity in B. rapa, despite significant differences in the kinetic and amplitude of the early signaling events. Our data support the relevance of PTI for crop protection and highlight specific functional target for disease resistance breeding in Brassica crops.

Host adaptation and microbial competition drive Ralstonia solanacearum phylotype I evolution in the Republic of Korea.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I (R. pseudosolanacearum) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role in Ralstonia evolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.

An immune receptor complex evolved in soybean to perceive a polymorphic bacterial flagellin.

In both animals and plants, the perception of bacterial flagella by immune receptors elicits the activation of defence responses. Most plants are able to perceive the highly conserved epitope flg22 from flagellin, the main flagellar protein, from most bacterial species. However, flagellin from Ralstonia solanacearum, the causal agent of the bacterial wilt disease, presents a polymorphic flg22 sequence (flg22Rso) that avoids perception by all plants studied to date. In this work, we show that soybean has developed polymorphic versions of the flg22 receptors that are able to perceive flg22Rso. Furthermore, we identify key residues responsible for both the evasion of perception by flg22Rso in Arabidopsis and the gain of perception by the soybean receptors. Heterologous expression of the soybean flg22 receptors in susceptible plant species, such as tomato, enhances resistance to bacterial wilt disease, demonstrating the potential of these receptors to enhance disease resistance in crop plants.

Erratum: Ralstonia solanacearum Type III Effectors with Predicted Nuclear Localization Signal Localize to Various Cell Compartments and Modulate Immune Responses in Nicotiana spp.

[This corrects the article on p. 43 in vol. 36, PMID: 32089660.].

Ralstonia solanacearum Type III Effectors with Predicted Nuclear Localization Signal Localize to Various Cell Compartments and Modulate Immune Responses in Nicotiana spp.

Ralstonia solanacearum (Rso) is a causal agent of bacterial wilt in Solanaceae crops worldwide including Republic of Korea. Rso virulence predominantly relies on type III secreted effectors (T3Es). However, only a handful of Rso T3Es have been characterized. In this study, we investigated subcellular localization of and manipulation of plant immunity by 8 Rso T3Es predicted to harbor a nuclear localization signal (NLS). While 2 of these T3Es elicited cell death in both Nicotiana benthamiana and N. tabacum, only one was dependent on suppressor of G2 allele of skp1 (SGT1), a molecular chaperone of nucleotide-binding and leucine-rich repeat immune receptors. We also identified T3Es that differentially regulate flg22-induced reactive oxygen species production and gene expression. Interestingly, several of the NLS-containing T3Es translationally fused with yellow fluorescent protein accumulated in subcellular compartments other than the cell nucleus. Our findings bring new clues to decipher Rso T3E function in planta.

Whole Genome Enabled Phylogenetic and Secretome Analyses of Two Venturia nashicola Isolates.

Venturia nashicola is a fungal pathogen causing scab disease in Asian pears. It is particularly important in the Northeast Asia region where Asian pears are intensively grown. Venturia nashicola causes disease in Asian pear but not in European pear. Due to the highly restricted host range of Venturia nashicola, it is hypothesized that the small secreted proteins deployed by the pathogen are responsible for the host determination. Here we report the whole genome based phylogenetic analysis and predicted secretomes for V. nashicola isolates. We believe that our data will provide a valuable information for further validation and functional characterization of host determinants in V. nashicola.

Modulation of plant innate immune signaling by small peptides.

Small peptides regulate the cellular coordination of growth, development, and stress tolerance in plants. In addition to direct antimicrobial activities, small secreted peptides have emerged as key signaling molecules in the plant immune response. Here, we highlight recent discoveries of several small peptides that amplify and fine-tune immune signaling.

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.