Plant-Microbe Interactions: Pathogenesis & Symbiosis
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Dissecting the virulence‐related functionality and cellular transcription mechanism of a conserved hypothetical protein in Xanthomonas oryzae pv. oryzae

Hypothetical proteins without defined functions are largely distributed in all sequenced bacterial genomes. Understanding their potent functionalities is a basic demand for bacteriologists. Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight of rice, is one of the model systems for studying molecular plant pathology. One quarter of proteins in the genome of this bacterium were defined as hypothetical proteins, but their roles in Xoo pathogenicity is unknown. Here, we generated in-frame deletions for six hypothetical proteins selected from strain PXO99A and found one (PXO_03177) of them is required for the full virulence of this strain. PXO_03177 is conserved in Xanthomonas, and is predicted to contain two domains relating to polysaccharide synthesis. However, we found that mutation of this gene did not affect the production or modification of extracellular polysaccharides (EPS) and lipopolysaccharides (LPS), two major polysaccharides produced by Xoo relating to its infection. Interestingly, we found that inactivation of PXO_03177 significantly impaired the biofilm formation and tolerance to SDS, all of which are considered to play key roles during the infection of Xoo on rice leaves. These findings thus enable us to define a function for PXO_03177 in the virulence of Xoo. Furthermore, we also found that the global regulator Clp controls the transcription of PXO_03177 by directly binding to its promoter region, presenting the first cellular regulatory pathway for modulating expression of this hypothetical protein gene. Our results provide the respective reference information for PXO_03177 homologues in Xanthomonas. This article is protected by copyright. All rights reserved.
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Frontiers | Roots and nodules response differently to P starvation in the Mediterranean-type legume Virgilia divaricata | Plant Science

Virgilia divaricata is a tree legume that grows in the Cape Floristic Region (CFR) in poor soils. A comparison between high and low phosphate growth conditions between roots and nodules was conducted and evaluated for the plants ability to handle low phosphate (LP) stress conditions in Virgilia divaricata. We showed that the plant copes with low phosphate stress through an increased allocation of resources, reliance on biological nitrogen fixation (BNF) and enhanced enzyme activity, especially phosphoenolpyruvate carboxylase . Nodules had a lower percentage decline in P compared to roots to uphold their metabolic functions. These strategies partly explain how V. divaricata can sustain growth despite LP conditions. Although the number of nodules declined with LP (i.e., 34% compared to the 88% decline in roots), their biomass remained unchanged in spite of an overall decline in plant dry weight. We attribute this behaviour to P conservation strategies in LP nodules that imply an increase in a metabolic bypass that operates at the phosphoenolpyruvate branchpoint in glycolysis. The enhanced activities of nodule phosphoenolpyruvate carboxylase, malate dehydrogenase and malic enzime, whilst pyruvate kinase declines, suggests that under LP conditions an adenylate bypass was in operation either to synthesize more organic acids or to mediate pyruvate via a non-adenylate requiring metabolic route. Both possibilities represent a P-stress adaptation route and this is the first report of its kind for legume trees that are indigenous to low P, acid soils. Although BNF declined by a small percentage during LP, this P conservation was evident in the unchanged BNF efficiency per weight, and the increase in BNF efficiency per mol of P. It appears that legumes that are indigenous to acid soils, may be able to continue their reliance on BNF via increased allocation to nodules and also due to increase their efficiency for BNF on a P basis, owing to P-saving mechanisms such as the organic acid routes.
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The Identity of a Single Residue of the RNA-dependent RNA Polymerase of Grapevine Fanleaf Virus Modulates Vein Clearing Symptoms in Nicotiana benthamiana | Molecular Plant-Microbe Interactions

The mechanisms underlying host plant symptom development upon infection by viruses of the genus Nepovirus in the family Secoviridae, including grapevine fanleaf virus (GFLV), are poorly understood. In the systemic host Nicotiana benthamiana, GFLV strain GHu produces characteristic vein clearing symptoms in apical leaves unlike other GFLV strains, such as F13, which cause an asymptomatic infection. In this study, we expanded on earlier findings and used reverse genetics to identify residue 802 (lysine, K) of the GFLV-GHu RNA1-encoded RNA-dependent RNA polymerase (1EPol) as a modulator of vein clearing symptom development in N. benthamiana. Mutations to this site abolished (K to G, A, or Q) or attenuated (K to N or P) symptom expression. Noteworthy, residue 802 is necessary but not sufficient for vein clearing symptoms as GFLV-F13 RNA1 carrying K802 remained asymptomatic in N. benthamiana. No correlation was found between symptom expression and RNA1 accumulation, as shown by RT-qPCR. Additionally, the involvement of RNA silencing in vein clearing symptoms was ruled out by VIGS experiments and structure predictions for protein 1EPol suggested that residue 802 is flanked by strongly predicted stable secondary structures, including a conserved motif of unknown function (805LLKT/AHLK/RT/ALR814). Together, these results reveal the protein nature of the GFLV-GHu symptom determinant in N. benthamiana and provide a solid basis for probing and determining the virus-host proteome network for vein clearing symptoms.
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Frontiers | Lipid Trafficking at Membrane Contact Sites During Plant Development and Stress Response | Plant Science

Frontiers | Lipid Trafficking at Membrane Contact Sites During Plant Development and Stress Response | Plant Science | Plant-Microbe Interactions: Pathogenesis & Symbiosis | Scoop.it
The biogenesis of cellular membranes involves an important traffic of lipids from their site of synthesis to their final destination. Lipid transfer can be mediated by vesicular or non-vesicular pathways. The non-vesicular pathway requires the close apposition of two membranes to form a functional platform, called membrane contact sites (MCSs), where lipids are exchanged. These last decades, MCSs have been observed between virtually all organelles and a role in lipid transfer has been demonstrated for some of them. In plants, the lipid composition of membranes is highly dynamic and can be drastically modified in response to environmental changes. This highlights the importance of understanding the mechanisms involved in the regulation of membrane lipid homeostasis in plants. This review summarizes our current knowledge about the non-vesicular transport of lipids at MCSs in plants and its regulation during stress.
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Foliar fungi alter reproductive timing and allocation in Arabidopsis under normal and water-stressed conditions

Microbes influence plant phenotypes but most known examples of this are from the study of below-ground microbes and plant disease modification. To examine the potential importance of phyllosphere microbes on non-disease related plant traits, we used sterile Arabidopsis clones to test the effects of foliar fungi on flowering phenology and reproductive allocation under conditions of varying water stress. We inoculated the sterile plants with fully-factorial combinations of four fungal isolates, then measured flowering time and reproductive allocation for each treatment group under normal and water-stressed conditions. All plants inoculated with foliar fungi had significantly later flowering and greater seed mass than the sterile control groups. The magnitude of this effect depended on the specific fungi present, but individual fungal effects diminished as inoculum richness increased. Above-ground microbes likely influence other plant traits as well and should be considered in any study measuring plant phenotypes.
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Selection of reference genes for quantitative real-time PCR normalization in the plant pathogen Puccinia helianthi Schw. | BMC Plant Biology | Full Text

Real-time RT-PCR has become a common and robust technique to detect and quantify low-abundance mRNA expression and is a prefered tool when examining fungal gene expression in infected host tissues. However, correct evaluation of gene expression data requires accurate and reliable normalization against a reference transcript. Thus, the identification of reference genes with stable expression during different conditions is of paramount importance. Here, we present a study where in vitro and in planta experiments were used to validate the expression stability of reference gene candidates of Puccinia helianthi Schw., an obligate pathogen that causes rust in sunflower (Helianthus annuus).

Results
Eleven reference genes of P. helianthi were validated at different growth stages. Excel-based software geNorm, BestKeeper and NormFinder were used to evaluate the reference gene transcript stabilities. Of eleven reference gene candidates tested, three were stably expressed in urediniospores, germinating growth stage and in planta. Two of these genes (UBC, EF2), encoding ubiquitin-conjugating enzyme and elongation factor 2, proved to be the most stable set of reference genes under the experimental conditions used.

Conclusion
We found that UBC and EF2 are suitable candidates for for the standardization of gene expression studies in the plant pathogen P. helianthi and potentially other related pathogens.
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A Plant Immune Receptor Adopts a Two-Step Recognition Mechanism to Enhance Viral Effector Perception

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen surveillance. Most plant NLRs characterized so far were found to use single domain/sensor to recognize pathogen effectors. Here we report that Sw-5b NLR immune receptor uses two distinct domains to detect the viral movement protein NSm encoded by tospovirus. In addition to the previously reported LRR domain, we found that the N-terminal Solanaceae domain (SD) of Sw-5b also interacts with NSm and a conserved 21-amino acids region of NSm (NSm21). The specific interaction between Sw-5b SD and NSm is required for releasing the CC inhibition on NB-ARC-LRR region. Our results showed that the binding of NSm affects the nucleotide-binding activity of the NB-ARC-LRR in vitro. However, Sw-5b NB-ARC-LRR is activated only when NSm and NSm21 levels were higher. Strikingly, Sw-5b SD could significantly enhance the ability of NB-ARC-LRR to detect low levels of NSm effector and facilitate its activation to induce defense response. The Sw-5b SD mutant that is disrupted in NSm recognition failed to enhance the ability of NB-ARC-LRR in sensing low levels of NSm and NSm21. Taken together, our results suggest that Sw-5b SD functions as an extra sensor and the NB-ARC-LRR functions as an activator and Sw-5b NLR adopts a two-step recognition mechanism to enhance viral effector perception.
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Exploring the role of ectomycorrhizal fungi in soil carbon dynamics - Zak - - New Phytologist - Wiley Online Library

The extent to which ectomycorrhizal fungi (ECM) enable plants to access organic nitrogen (N) bound in soil organic matter (SOM) and transfer this growth‐limiting nutrient to their plant host, has important implications for our understanding of plant–fungal interactions and the cycling and storage of carbon (C) and N in terrestrial ecosystems. Empirical evidence currently supports a range of perspectives, suggesting that ECM vary in their ability to provide their host with N bound in SOM, and that this capacity can both positively and negatively influence soil C storage. To help resolve the multiplicity of observations, we gathered a group of researchers to explore the role of ECM fungi in soil C dynamics, and propose new directions that hold promise to resolve competing hypotheses and contrasting observations. In this Viewpoint, we summarize these deliberations and identify areas of inquiry that hold promise for increasing our understanding of these fundamental and widespread plant symbionts and their role in ecosystem level biogeochemistry.
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Osa‐miR398b boosts H2O2 production and rice blast disease‐resistance via multiple superoxide dismutases - Li - - New Phytologist - Wiley Online Library

miRNAs contribute to plant resistance against pathogens. Previously, we found that the function of miR398b in immunity in rice differs from that in Arabidopsis. However, the underlying mechanisms are unclear.

In this study, we characterized the mutants of miR398b target genes and demonstrated that multiple superoxide dismutase genes contribute to miR398b‐regulated rice immunity against the blast fungus Magnaporthe oryzae.

Out of the four target genes of miR398b, mutations in Cu/Zn‐Superoxidase Dismutase1 (CSD1), CSD2 and Os11g09780 (SODX) led to enhanced resistance to M. oryzae and increased H2O2 accumulation. By contrast, mutations in Copper Chaperone for Superoxide Dismutase (CCSD) resulted in enhanced susceptibility. Biochemical studies revealed that csd1, csd2 and sodx displayed altered expression of CSDs and other SOD family members, leading to increased total SOD enzyme activity that positively contributed to higher H2O2 production. In contrast, the ccsd mutant showed CSD protein deletion, resulting in decreased CSD and total SOD enzyme activity.

Our results demonstrate the roles of different SODs in miR398b‐regulated resistance to rice blast disease, and uncover an integrative regulatory network in which miR398b boosts total SOD activity to up‐regulate H2O2 level and thereby improve disease resistance.
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A novel fungal effector from Puccinia graminis suppressing RNA silencing and plant defense responses - Yin - - New Phytologist - Wiley Online Library

Fungal plant pathogens like rust‐causing biotrophic fungi secrete hundreds of effectors into plant cells to subvert host immunity and promote pathogenicity on their host plants by manipulating specific physiological processes or signal pathways, but the actual function has been demonstrated for very few of these proteins.

Here, we show that the PgtSR1 effector proteins, encoded by two allelic genes (PgtSR1‐a and PgtSR1‐b), from the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt), suppress RNA silencing in plants and impede plant defenses by altering the abundance of small RNAs that serve as defense regulators.

Expression of the PgtSR1s in plants revealed that the PgtSR1s promote susceptibility to multiple pathogens and partially suppress cell death triggered by multiple R proteins.

Overall, our study provides the first evidence that the filamentous fungus Puccinia graminis has evolved to produce fungal suppressors of RNA silencing (FSR) and indicates that PgtSR1s suppress both basal defenses and effector triggered immunity.
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OsSYP121 accumulates at fungi penetration sites and mediates host resistance to rice blast

Magnaporthe oryzae is a fungal pathogen that causes rice (Oryza sativa) blast. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells and are known to contribute to fungal pathogen resistance. Syntaxin of Plants 121 (SYP121), a Qa-SNARE, has been reported to function in non-host resistance in Arabidopsis thaliana. However, the functions of SYP121 in host resistance to rice blast are largely unknown. Here we report that the rice SYP121 protein, OsSYP121, accumulates at fungal penetration sites and mediates host resistance to rice blast. OsSYP121 is plasma membrane-localized and its expression was obviously induced by the rice blast in both the blast-resistant rice landrace Hei (Heikezijing) and the blast-susceptible landrace Su (Suyunuo). Overexpression of OsSYP121 in Su resulted in enhanced resistance to blast. Knockdown of OsSYP121 expression in Su resulted in a more susceptible phenotype. However, knockdown of OsSYP121 expression in the resistant cultivar Hei resulted in susceptibility to the blast fungus. The POsSYP121::GFP-OsSYP121 accumulated at rice blast penetration sites in transgenic rice, as observed by confocal microscopy. Yeast two-hybrid results showed that OsSYP121 can interact with OsSNAP32 (Synaptosome-associated protein of 32 kDa) and OsVAMP714/724 (Vesicle-associated membrane protein714/724). The interaction between OsSYP121 and OsSNAP32 may contribute to host resistance to rice blast. Our study reveals that OsSYP121 plays an important role in rice blast resistance as it is a key component in vesicle trafficking.
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MYC2 Regulates the Termination of Jasmonate Signaling via an Autoregulatory Negative Feedback Loop

In tomato (Solanum lycopersicum), as with in other plants, the immunity hormone jasmonate (JA) triggers genome-wide transcriptional changes in response to pathogen and insect attack. These changes are largely regulated by the basic helix-loop-helix (bHLH) transcription factor MYC2. The function of MYC2 depends on its physical interaction with the MED25 subunit of the Mediator transcriptional co-activator complex. Although much has been learned about the MYC2-dependent transcriptional activation of JA-responsive genes, relatively less studied is the termination of JA-mediated transcriptional responses and the underlying mechanisms. Here, we report an unexpected function of MYC2 in regulating the termination of JA signaling through activating a small group of JA-inducible bHLH proteins, termed MYC2-TARGETED BHLH 1 (MTB1), MTB2, and MTB3. MTB proteins negatively regulate JA-mediated transcriptional responses via their antagonistic effects on the functionality of the MYC2-MED25 transcriptional activation complex. MTB proteins impair the formation of the MYC2-MED25 complex and compete with MYC2 to bind to its target gene promoters. Therefore, MYC2 and MTB proteins form an autoregulatory negative feedback circuit to terminate JA signaling in a highly organized manner. We exemplify that new gene editing tools, such as CRISPR/Cas9, open up new avenues to exploit MTB genes for crop protection.
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Identification and characterisation of hypomethylated DNA loci controlling quantitative resistance in Arabidopsis

Variation in DNA methylation enables plants to inherit traits independently of changes to DNA sequence. Here, we have screened an Arabidopsis population of epigenetic recombinant inbred lines (epiRILs) for resistance against Hyaloperonospora arabidopsidis (Hpa). These lines share the same genetic background, but show variation in heritable patterns of DNA methylation. We identified 4 epigenetic quantitative trait loci (epiQTLs) that provide quantitative resistance without reducing plant growth or resistance to other (a)biotic stresses. Phenotypic characterisation and RNA-sequencing analysis revealed that Hpa-resistant epiRILs are primed to activate defence responses at the relatively early stages of infection. Collectively, our results show that hypomethylation at selected pericentromeric regions is sufficient to provide quantitative disease resistance, which is associated with genome-wide priming of defence-related genes. Based on comparisons of global gene expression and DNA methylation between the wild-type and resistant epiRILs, we discuss mechanisms by which the pericentromeric epiQTLs could regulate the defence-related transcriptome.
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An EDS1-SAG101 complex functions in TNL-mediated immunity in Solanaceae

EDS1 (Enhanced disease susceptibility 1) forms mutually exclusive heterodimers with its interaction partners PAD4 (Phytoalexin-deficient 4) and SAG101 (Sensecence-associated gene 101). Collectively, these complexes are required for resistance responses mediated by nucleotide-binding leucine-rich repeat-type immune receptors (NLRs) possessing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs). Here, immune functions of EDS1 complexes were comparatively analyzed in a mixed species approach relying on Nicotiana benthamiana (Nb), Solanum lycopersicum (Sl) and Arabidopsis thaliana (At). Genomes of most Solanaceae plants including Nb and Sl encode for two SAG101 isoforms, which engage into distinct complexes with EDS1. By a combination of genome editing and transient complementation, we show that one of these EDS1-SAG101 complexes, and not an EDS1-PAD4 complex as previously described in At, is necessary and sufficient for all tested TNL-mediated immune responses in Nb. Intriguingly, not this EDS1-SAG101 module, but mainly Solanaceae EDS1-PAD4 execute immune functions when transferred to At, and TNL functions are not restored in Nb mutant lines by expression of At EDS1 complexes. We conclude that EDS1 complexes do not represent a complete functional module, but co-evolve with additional factors, most likely protein interaction partners, for their function in TNL signaling networks of individual species. In agreement, we identify a large surface on SlEDS1 complexes required for immune activities, which may function in partner recruitment. We highlight important differences in TNL signaling networks between At and Nb, and genetic resources in the Nb system will be instrumental for future elucidation of EDS1 molecular functions.
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1-aminocyclopropane-1-carboxylate: a novel and strong chemoattractant for the plant beneficial rhizobacterium Pseudomonas putida UW4 | Molecular Plant-Microbe Interactions

PGPR and fungi-bacterial biofilms are both important biofertilizer inoculants for sustainable agriculture. However, the strongest chemoattractant for bacteria to colonize the rhizosphere and mycelia is not clear. Coincidentally, almost all of the PGPRs possess 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AcdS) and can utilize ACC as the sole nitrogen source. Here, we found that ACC was a novel, metabolic dependent and methyl-accepting chemoreceptor (MCP)-involved chemoattractant for Pseudomonas putida UW4. The chemotactic response of UW4 to ACC is significantly greater than that to the amino acids and organic acids identified in the plant root and fungal hyphal exudates. The colonization accounts of the acdS or cheR deletion mutants of UW4 in the wheat rhizosphere and on Agaricus bisporus mycelia were reduced one magnitude compared to those of UW4. The colonization accounts of UW4 on A. bisporus antisense ACO mycelia with a high ACC production significantly increased compared to A. bisporus, following by UW4 cheR complementary strain and ethylene chemoreceptor gene deletion mutant. The colonization accounts of the UW4 strains on A. bisporus acdS+ mycelia with a low ACC production decreased significantly compared to A. bisporus wt. The results suggested that ACC, not ethylene, should be the strongest chemoattractant for the PGPR that contain AcdS.
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Biocontrol of soft-rot: confocal microscopy highlights virulent pectobacterial communication and its jamming by rhodococcal quorum-quenching | Molecular Plant-Microbe Interactions

Confocal laser scanning microscopy was chosen to observe the colonization and damage caused by the soft-rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection and/or degradation. The dual-colored pathogen and biocontrol strains were used to co-inoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls as well as a concomitant rhodococcal quorum-quenching response, both at the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.
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Comparative transcriptomic analysis reveals different responses of Arabidopsis thaliana roots and shoots to infection by Agrobacterium tumefaciens in a hydroponic co-cultivation system - ScienceDirect

Comparative transcriptomic analysis reveals different responses of Arabidopsis thaliana roots and shoots to infection by Agrobacterium tumefaciens in a hydroponic co-cultivation system - ScienceDirect | Plant-Microbe Interactions: Pathogenesis & Symbiosis | Scoop.it
Agrobacterium tumefaciens-Arabidopsis thaliana provides an excellent model system to study bacterial pathogenesis and plant interactions. It is well established that, upon recognizing plant-derived signals, A. tumefaciens activates its virulence programing, culminated by the transfer and integration of T-DNA into the plant genome. However, the initial signaling and response of the plant host during A. tumefaciens -plant interactions is less understood. In this study, A. thaliana were co-cultivated (infected) with A. tumefaciens in a sterile hydroponic system for 8 h, and the transcriptome profiles of root and shoot were determined by microarray analysis. Overall, hundreds of genes involved in plant responses to stress, regulation of transcription, signal transduction or plant metabolism are differentially expressed in roots and shoots upon A. tumefaciens infection. In total, 323 genes were up-regulated and 226 were down-regulated by a log2 transformed fold change of at least 2 in roots upon A. tumefaciens infection, as compared with mock-inoculated plants. In the shoot, 249 genes were up-regulated and 152 were down-regulated in response to A. tumefaciens infection in the roots. Functional classification and comparative analysis revealed that, in response to pathogen attack, the host plant demonstrated significantly differential signaling loops between locally (roots) and distally (shoots) affected sites, despite of little overlap between transcriptome responses. Upon A. tumefaciens infection, roots displayed a strong induction of defense and hormone signaling pathways involving salicylic acid, abscisic acid, ethylene, and auxins, which play essential roles in plant acclimation responses in the root. On the contrary, jasmonic acid and gibberellic acid signaling processes were most activated in the shoots. The identification of genes and signaling pathways that are differentially regulated in roots and shoots may serve as possible targets in molecular breeding for disease tolerant plants or for improving Agrobacterium-mediated plant transformation.
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feedback loop between CaWRKY41 and H2O2 coordinates pepper’s response to Ralstonia solanacearum and excess cadmium | Journal of Experimental Botany | Oxford Academic

WRKY transcription factors have been implicated in both plant immunity and plant responses to cadmium (Cd); however, the mechanism underlying the crosstalk between these processes is unclear. Here, we characterized the roles of CaWRKY41, a groupⅢ WRKY TF, in immunity against the pathogenic bacterium Ralstonia solanacearum and Cd stress responses in pepper (Capsicum annuum). CaWRKY41 was transcriptionally upregulated in response to Cd exposure, R. solanacearum inoculation, and H2O2 treatment. Virus-induced silencing of CaWRKY41 increased Cd tolerance and R. solanacearum susceptibility, while heterologous overexpression of CaWRKY41 in Arabidopsis impaired Cd tolerance, and enhanced Cd and Zn uptake and H2O2 accumulation. Genes encoding reactive oxygen species (ROS)-scavenging enzymes were downregulated in overexpressing CaWRKY41Arabidopsis plants, whereas those encoding Zn transporters and enzymes involved in H2O2 production were upregulated. Consistent with these findings, the ocp3 (overexpressor of cationic peroxidase 3) mutant, which has elevated H2O2 levels, displayed enhanced sensitivity to Cd stress. These results suggest that a positive feedback loop between H2O2 accumulation and CaWRKY41 upregulation coordinates the responses of pepper to R. solanacearum inoculation and Cd exposure. This mechanism might reduce Cd tolerance by increasing Cd uptake via Zn transporters, while enhancing resistance to R. solanacearum.
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Rice black‐streaked dwarf virus P10 acts as either a synergistic or an antagonistic determinant during superinfection with related or unrelated virus - Zhang - - Molecular Plant Pathology - Wiley O...

Rice black‐streaked dwarf virus (RBSDV), a member of the genus Fijivirus, is a devastating pathogen of crop plants. RBSDV S10 encodes a capsid protein (P10) that is an important component of the double‐layered particle. However, little information is available on the roles of RBSDV P10 in viral infection or in interactions with other viruses. Here we demonstrate that expressing P10 in plants alleviated the symptoms of both RBSDV and the closely‐related southern rice black‐streaked dwarf virus (SRBSDV), and reduced the disease incidence, but made the plants more susceptible to the unrelated rice stripe virus (RSV). Further experiments suggested that P10‐mediated resistance to RBSDV and SRBSDV operated at the protein level rather than the RNA level and was not due to post transcriptional gene silencing. Transcriptomic data revealed that expressing P10 in plants significantly suppressed the expression of rice defense‐related genes, which might play important roles in resistance to RSV infection. After infection with RBSDV, plants were more resistant to subsequent challenge by SRBSDV but more susceptible to RSV. Overall, these results indicate that P10 acts as an important effector in virus interactions.
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Inside out: root cortex‐localized LHK1 cytokinin receptor limits epidermal infection of Lotus japonicus roots by Mesorhizobium loti - Miri - - New Phytologist - Wiley Online Library

During Lotus japonicas–Mesorhizobium loti symbiosis, the LOTUS HISTIDINE KINASE1 (LHK1) cytokinin receptor regulates both the initiation of nodule formation and the scope of root infection. However, the exact spatio‐temporal mechanism by which this receptor exerts its symbiotic functions has remained elusive.

•In this study, we performed cell type‐specific complementation experiments in the hyperinfected lhk1‐1 mutant background, targeting LHK1 to either the root epidermis or the root cortex. We also utilized various genetic backgrounds to characterize expression of several genes regulating symbiotic infection.

•We show here that expression of LHK1 in the root cortex is required and sufficient to regulate both nodule formation and epidermal infections. The LHK1‐dependent signalling that restricts subsequent infection events is triggered before initial cell divisions for nodule primordium formation. We demonstrate also that AHK4, the Arabidopsis orthologue of LHK1, is able to regulate M. loti infection in L. japonicus, suggesting that an endogenous cytokinin receptor could be sufficient for engineering nitrogen‐fixing root nodule symbiosis in nonlegumes.

•Our data provide experimental evidence for the existence of an LHK1‐dependent root cortex‐to‐epidermis feedback mechanism regulating rhizobial infection. This root‐localized regulatory module functionally links with the systemic autoregulation of nodulation (AON) to maintain the homeostasis of symbiotic infection.
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Bacterially produced spermidine induces plant systemic susceptibility to pathogens

Plant root-associated microbes promote plant growth, in part by the induction of systemic resistance (ISR) to foliar pathogens. In an attempt to find novel growth-promoting and ISR inducing strains, we previously identified strains of root-associated Pseudomonas spp. that promote plant growth but unexpectedly induced systemic susceptibility (ISS) to foliar pathogens. Here we demonstrate that the ISS-inducing phenotype is common among root-associated Pseudomonas spp. and we identified the underlying genetic and molecular basis of ISS. Using comparative genomics we identified a single P. fluorescens locus containing a novel periplasmic spermidine biosynthesis gene speE2 that is unique to ISS strains. We generated a clean deletion of the speE2 gene in two ISS strains and found that speE2 is necessary for the ISS phenotype. Spermidine but not spermine is sufficient to phenocopy ISS strains. The ISS locus is present in diverse bacteria and has previously been implicated in pathogenesis in animals. Collectively these data show that a single bacterially derived molecule can modulate systemic plant immunity.
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Chloroplasts navigate towards the pathogen interface to counteract infection by the Irish potato famine pathogen

Chloroplasts are light harvesting organelles that arose from ancient endosymbiotic cyanobacteria. Upon immune activation, chloroplasts switch off photosynthesis, produce anti-microbial compounds, and develop tubular extensions called stromules. We report that chloroplasts navigate to the pathogen interface to counteract infection by the Irish potato famine pathogen Phytophthora infestans, physically associating with the specialised membrane that engulfs pathogen haustoria. Outer envelope protein, chloroplast unusual positioning1 (CHUP1), anchors chloroplasts to the host-pathogen interface. Stromules are induced during infection in a CHUP1-dependent manner, embracing haustoria and interconnecting chloroplasts, to form dynamic organelle clusters. Infection-triggered reprogramming of chloroplasts relies on surface immune signalling, whereas pathogen effectors subvert these immune pulses. Chloroplast are deployed focally, and coordinate to restrict pathogen entry into plant cells, a process actively countered by parasite effectors.
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A small cysteine‐rich protein from two kingdoms of microbes is recognized as a novel pathogen‐associated molecular pattern - Nie - - New Phytologist - Wiley Online Library

Summary
Pathogen‐associated molecular patterns (PAMPs) are conserved molecules that are crucial for normal life cycle of microorganisms. However, the diversity of microbial PAMPs is little known. During screening of cell‐death‐inducing factors from the necrotrophic fungus Valsa mali, we identified a novel PAMP VmE02 that is widely spread in oomycetes and fungi.
Agrobacterium tumefaciens‐mediated transient expression or infiltration of recombinant protein produced by Escherichia coli was performed to assay elicitor activity of the proteins tested. Virus‐induced gene silencing in Nicotiana benthamiana was used to determine the components involved in VmE02‐triggered cell death. The role of VmE02 in virulence and conidiation of V. mali were characterized by gene deletion and complementation.
We found that VmE02, together with some of its homologues from both oomycete and fungal species, exhibited cell‐death‐inducing activity in N. benthamiana. VmE02‐triggered cell death was shown to be dependent on BRI1‐ASSOCIATED KINASE‐1, SUPPRESSOR OF BIR1‐1, HSP90 and SGT1 in N. benthamiana. Deletion of VmE02 in V. mali greatly attenuated pathogen conidiation but not virulence, and treatment of N. benthamiana with VmE02 enhances plant resistance to Sclerotinia sclerotiorum and Phytophthora capsici.
We conclude that VmE02 is a novel cross‐kingdom PAMP produced by several fungi and oomycetes.
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Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae - Zhang - - New Phytologist - Wiley Online Library

Verticillium dahliae is a broad host‐range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved.

Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis and quantification of V. dahliae secondary metabolites were performed.

Population genomics previously revealed that G‐LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Seven gene deletion mutants within G‐LSR2, designated as VdDfs, produced a nondefoliating phenotype on cotton, olive, and okra. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, while VdDf7 shared homology with proteins involved in the biosynthesis of N‐lauroylethanolamine (NAE 12:0), a compound that induces defoliation. NAE over‐biosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase.

The VdDfs modulate the synthesis and over‐production of secondary metabolites such as NAE 12:0 that causes defoliation either by altering abscisic acid (ABA) sensitivity, hormone disruption, or sensitivity to the pathogen.
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Binding of the Magnaporthe oryzae chitinase MoChia1 by a rice tetratricopeptide repeat protein allows free chitin to trigger immune responses

To defend against pathogens, plants have developed complex immune systems, including plasma membrane receptors that recognize pathogen-associated molecular patterns, such as chitin from fungal cell walls, and mount a defense response. Here, we identify a chitinase, MoChia1, secreted by Magnaporthe oryzae, a fungal pathogen of rice (Oryza sativa). MoChia1 can trigger plant defense responses and expression of MoChia1 under an inducible promoter in rice enhances its resistance to M. oryzae. MoChia1 is a functional chitinase required for M. oryzae growth and development; knocking out MoChia1 significantly reduced the virulence of the fungus and we found that MoChia1 binds chitin to suppress the chitin-triggered plant immune response. However, the rice tetratricopeptide repeat protein OsTPR1 interacts with MoChia1 in the rice apoplast. OsTPR1 competitively binds MoChia1, thereby allowing the accumulation of free chitin and re-establishing the immune response. Overexpressing OsTPR1 in rice plants resulted in elevated levels of reactive oxygen species during M. oryzae infection. Our data demonstrate that rice plants not only recognize MoChia1, but also employ an OsTPR protein to counteract the function of this fungal chitinase and regain immunity.
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Role of MPK4 in pathogen-associated molecular pattern-triggered alternative splicing in Arabidopsis

Alternative splicing (AS) of pre-mRNAs in plants is an important mechanism of gene regulation in environmental stress tolerance but plant signals involved are essentially unknown. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is mediated by mitogen-activated protein kinases and the majority of PTI defense genes are regulated by MPK3, MPK4 and MPK6. These responses have been mainly analyzed at the transcriptional level, however many splicing factors are direct targets of MAPKs. Here, we studied alternative splicing induced by the PAMP flagellin in Arabidopsis. We identified 506 PAMP-induced differentially alternatively spliced (DAS) genes. Although many DAS genes are targets of nonsense-mediated degradation (NMD), only 19% are potential NMD targets. Importantly, of the 506 PAMP-induced DAS genes, only 89 overlap with the set of 1849 PAMP-induced differentially expressed genes (DEG), indicating that transcriptome analysis does not identify most DAS events. Global DAS analysis of mpk3, mpk4, and mpk6 mutants revealed that MPK4 is a key regulator of PAMP-induced differential splicing, regulating AS of a number of splicing factors and immunity-related protein kinases, such as the calcium-dependent protein kinase CPK28, the cysteine-rich receptor like kinases CRK13 and CRK29 or the FLS2 co-receptor SERK4/BKK1.These data suggest that MAP kinase regulation of splicing factors is a key mechanism in PAMP-induced AS regulation of PTI.
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