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Key message: The pathogenesis-related 1 gene of watermelon responds to the infection of ZYMV and contributes to the resistance of its host. Zucchini yellow mosaic virus (ZYMV; family Potyviridae) is a single-stranded positive-sense RNA virus that is a serious threat to cucurbits. Previously, we observed a hypersensitivity response (HR) in the systemic leaves of the 938-16-B watermelon line infected with ZYMV, distinct from the typical HR at infected sites. In this study, we confirmed that ZYMV accumulation in 938-16-B was significantly lower than in the susceptible line H1. Upon inoculation, the entry of ZYMV-eGFP into mesophyll cells is restricted into necrotic spots in leaves, indicating that resistance to ZYMV in 938-16-B is linked to the HR. Further, grafting experiments between 938-16-B and susceptible varieties were performed, and revealed an HR induction in susceptible varieties, suggesting the transfer of resistance signal(s) from 938-16-B to susceptible varieties. Through RNA-sequencing and proteomics analyses of the H1 scions on 938-16-B rootstock, a pathogenesis-related 1 (ClPR1) gene was identified. Specifically, ClPR1 expression is unique to ZYMV-infected 938-16-B. Repression of the expression of ClPR1 prevents an HR in 938-16-B. Conversely, overexpression of ClPR1 in susceptible varieties significantly reduces ZYMV accumulation, but an HR was not induced in susceptible line. Besides the virus, jasmonic acid (JA) can also trigger an HR in 938-16-B. Intriguingly, the expression of ClPR1 (Cla97C02G034020) is induced in both of 938-16-B and H1 by MeJA, rather than salicylic acid. These results suggest that HR is associated with the expression of ClPR1 and contributes to resistance to ZYMV in 938-16-B. [ABSTRACT FROM AUTHOR]

In plants, recognition between resistance gene (R) and virus induces a local hypersensitive response (HR), which is accompanied by systemic acquired resistance (SAR). The dominant resistance gene N in tobacco confers resistance to tobacco mosaic virus (TMV) at both locally inoculated tissues and systemically infected tissues. However, the mechanisms underlying HR- and SAR-mediated viral inhibition are not fully revealed. In this study, we find that Nicotiana glutinosa RNA-binding protein (NgRBP) is an RNA silencing suppressor which enhances TMV-triggered HR. Stronger HR could result in stronger local and systemic RNA silencing as well as SAR. Enhanced RNA silencing in the systemically infected leaves induced by the NgRBP gene is compromised by transient expression of NahG. These results indicate that RNA silencing is activated by HR and plays a crucial role in local and systemic virus resistance. Our results reveal a crosstalk between N gene-mediated virus resistance and RNA silencing which would deepen our understanding of the established HR and SAR models. [ABSTRACT FROM AUTHOR]

Abstract In plants, recognition between resistance gene (R) and virus induces a local hypersensitive response (HR), which is accompanied by systemic acquired resistance (SAR). The dominant resistance gene N in tobacco confers resistance to tobacco mosaic virus (TMV) at both locally inoculated tissues and systemically infected tissues. However, the mechanisms underlying HR- and SAR-mediated viral inhibition are not fully revealed. In this study, we find that Nicotiana glutinosa RNA-binding protein (NgRBP) is an RNA silencing suppressor which enhances TMV-triggered HR. Stronger HR could result in stronger local and systemic RNA silencing as well as SAR. Enhanced RNA silencing in the systemically infected leaves induced by the NgRBP gene is compromised by transient expression of NahG. These results indicate that RNA silencing is activated by HR and plays a crucial role in local and systemic virus resistance. Our results reveal a crosstalk between N gene-mediated virus resistance and RNA silencing which would deepen our understanding of the established HR and SAR models.

Blast disease, caused by the hemibiotrophic ascomycete fungus, Magnaporthe oryzae, is a significant threat to sustainable rice production worldwide. Studies have shown that the blast fungus secretes vast arrays of functionally diverse proteins into the host cell for a successful disease progression. However, the final destinations of these effector proteins inside the host cell and their role in advancing fungal pathogenesis remain a mystery. Here, we reported that a putative mitochondrial targeting non-classically secreted protein (MoMtp) positively regulates conidiogenesis and appressorium maturation in M. oryzae. Moreover, MoMTP gene deletion mutant strains triggered a hypersensitive response when inoculated on rice leaves displaying that MoMtp is essential for the virulence of M. oryzae. In addition, cell wall and oxidative stress results indicated that MoMtp is likely involved in the maintenance of the structural integrity of the fungus cell. Our study also demonstrates an upregulation in the expression pattern of the MoMTP gene at all stages of infection, indicating its possible regulatory role in host invasion and the infectious development of M. oryzae. Furthermore, Agrobacterium infiltration and sheath inoculation confirmed that MoMtp-GFP protein is predominantly localized in the host mitochondria of tobacco leaf and rice cells. Taken together, we conclude that MoMtp protein likely promotes the normal conidiation and pathogenesis of M. oryzae and might have a role in disturbing the proper functioning of the host mitochondria during pathogen invasion.

Movement proteins (MPs) encoded by plant viruses are essential for cell-to-cell transport of viral genomes through plasmodesmata. The genome of hibiscus green spot virus contains a module of two MP genes termed 'binary movement block' (BMB), encoding the proteins BMB1 and BMB2. Here, BMB1 is shown to induce a defense response in Nicotiana benthamiana plants that inhibits BMB-dependent virus transport. This response is characterized by the accumulation of reactive oxygen species, callose deposition in the cell wall, and upregulation of 9-LOX expression. However, the BMB1-induced response is inhibited by coexpression with BMB2. Furthermore, BMB1 is found to localize to subnuclear structures, in particular to Cajal bodies, in addition to the cytoplasm. As shown in experiments with a BMB1 mutant, the localization of BMB1 to nuclear substructures enhances BMB-dependent virus transport. Thus, the virus transport mediated by BMB proteins is modulated by (i) a BMB1-induced defense response that inhibits transport, (ii) suppression of the BMB1-induced response by BMB2, and (iii) the nuclear localization of BMB1 that promotes virus transport. Collectively, the data presented demonstrate multiple levels of interactions between viral pathogens and their plant hosts during virus cell-to-cell transport. [ABSTRACT FROM AUTHOR]

Harpins play a key role in inducing disease resistance in crops, and identifying their core functional regions and establishing a system for their efficient expression would be very valuable. In this study, large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions (28 ℃C with 0.5 mmol.L-1 IPTG for 6 h) in Escherichia coli BL21 (DE3). Hypersensitive response (HR) assays demonstrated that the C-terminal 66 aa of HrpZ (HrpZ_C_2_2) elicited a strong HR in tobacco (Nicotiana benthamiana) and grape (Flame Seedless) leaves. Additionally, treatment with HrpZ, and particularly HrpZ_C_2_2, significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew. The determination of the physiological parameters indicated that HrpZ, and especially HrpZ_C_2_2, improved the photosynthesis- and chlorophyll fluorescence-related parameters, enhanced the activity of defense-related enzymes, including SOD, POD, CAT and PAL, and increased the H2O2 level. Collectively, we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew. This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew. [ABSTRACT FROM AUTHOR]

SUMMARY: Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one‐carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens. Significance Statement: Arabidopsis photorespiratory SHMT6 plays a role in plant resistance. Overexpression of SHMT6 leads to enhanced production of ethylene and lignin, which are important components of the defense response. [ABSTRACT FROM AUTHOR]

This article comments on: Mutinda S, Mobegi FM, Hale B, Dayou O, Ateka E, Wijeratne A, Wicke S, Bellis ES, Runo S. 2023. Resolving intergenotypic Striga resistance in sorghum. Journal of Experimental Botany 74, 5294–5306.

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide–deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Accurate quantification of hypersensitive response (HR) programmed cell death is imperative for understanding plant defense mechanisms and developing disease‐resistant crop varieties. Here, a phenotyping platform for rapid, continuous‐time, and quantitative assessment of HR is demonstrated: Parallel Automated Spectroscopy Tool for Electrolyte Leakage (PASTEL). Compared to traditional HR assays, PASTEL significantly improves temporal resolution and has high sensitivity, facilitating detection of microscopic levels of cell death. Validation is performed by transiently expressing the effector protein AVRblb2 in transgenic Nicotiana benthamiana (expressing the corresponding resistance protein Rpi‐blb2) to reliably induce HR. Detection of cell death is achieved at microscopic intensities, where leaf tissue appears healthy to the naked eye one week after infiltration. PASTEL produces large amounts of frequency domain impedance data captured continuously. This data is used to develop supervised machine‐learning (ML) models for classification of HR. Input data (inclusive of the entire tested concentration range) is classified as HR‐positive or negative with 84.1% mean accuracy (F1 score = 0.75) at 1 h and with 87.8% mean accuracy (F1 score = 0.81) at 22 h. With PASTEL and the ML models produced in this work, it is possible to phenotype disease resistance in plants in hours instead of days to weeks. [ABSTRACT FROM AUTHOR]

P0 proteins encoded by the pepper vein yellow virus (PeVYV) are pathogenic factors that cause hypersensitive response (HR). However, the host gene expression related to PeVYV P0‐induced HR has not been thoroughly studied. Transcriptomic technology was used to investigate the host pathways mediated by the PeVYV P0 protein to explore the molecular mechanisms underlying its function. We found 12,638 differentially expressed genes (DEGs); 6784 and 5854 genes were significantly upregulated and downregulated, respectively. Transcriptomic and reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR) analyses revealed that salicylic acid (SA) and jasmonic acid (JA) synthesis‐related gene expression was upregulated, and ethylene synthesis‐related gene expression was downregulated. Ultrahigh performance liquid chromatography‐tandem mass spectrometry was used to quantify SA and JA concentrations in Nicotiana benthamiana, and the P0 protein induced SA and JA biosynthesis. We then hypothesized that the pathogenic activity of the P0 protein might be owing to proteins related to host hormones in the SA and JA pathways, modulating host resistance at different times. Viral gene silencing suppression technology was used in N. benthamiana to characterize candidate proteins, and downregulating NbHERC3 (Homologous to E6‐AP carboxy‐terminus domain and regulator of choromosome condensation‐1 dmain protein 3) accelerated cell necrosis in the host. The downregulation of NbCRR reduced cell death, while that of NbBax induced necrosis and curled heart leaves. Our findings indicate that NbHERC3, NbBax, and NbCRR are involved in P0 protein‐driven cell necrosis. [ABSTRACT FROM AUTHOR]

This article explores the role of the WAK3 gene in Arabidopsis plants' immune responses to harpins, proteins produced by certain bacteria that activate plant defenses. The researchers used T-DNA insertion mutants to study WAK3 and found that it is essential for the necrosis and acquired resistance responses triggered by harpins. They also discovered that genetic variation in harpin perception may impact plant fitness. The study benefited from the use of the 1001 Genomes data, which eliminated the need for lengthy genetic mapping. This research provides valuable insights into the genetic basis of plant immune responses and its potential implications for crop protection. [Extracted from the article]

Imbibing watermelon seeds in 1 mM sodium tetraborate (Na2B4O7) for 24 h systemically protected plants against foliar infection by Stagonosporopsis cucurbitacearum in detached leaves and under greenhouse conditions. The treatment resulted in both a reduction in the overall percentage of leaf infection as well as in the size of lesions. Studies of the mechanisms by which Na2B4O7 protected watermelon showed that there was no direct effect on the S. cucurbitacearum mycelium growth in vitro. On the other hand, plants raised from seeds primed with Na2B4O7 showed a higher frequency of fluorescent epidermal cells compared to the plants treated with water. This indicates that a higher number of cells expressed the hypersensitive response after Na2B4O7 priming. In addition, there was an increase in peroxidase activity and an enhanced accumulation of a 45 kDa acidic peroxidase isoform during the early stages of infection in plants treated with Na2B4O7 compared to plants treated with water and this was positively correlated to the reduction of leaf infection caused by the pathogen. These results indicate that Na2B4O7 is able to induce systemic resistance in watermelon against S. cucurbitacearum by activating the hypersensitive reaction at penetration sites, increasing peroxidase activity and altering the peroxidase isozyme profile. Although each individual response may only have had a minor effect, their combined effects had a reducing effect on the disease. [ABSTRACT FROM AUTHOR]

The basic region/leucine zipper (bZIP) transcription factors play important roles in plant development and responses to abiotic and biotic stresses. OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5-RNAi transgenic plants. To further investigate the biological functions of OsbZIP53, we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants. Comprehensive analysis of phenotypical, physiological, and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants, but also regulated the immune response through the salicylic acid pathway. Specifically, disrupting OsbZIP53 increased H2O2 accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs (Osrboh genes) and weakened H2O2 degradation by directly targeting OsMYBS1. In addition, the growth of osbzip53 mutants was seriously impaired, while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type, suggesting that OsbZIP53 has a balancing effect on rice immune response and growth.

The basic region/leucine zipper (bZIP) transcription factors play important roles in plant development and responses to abiotic and biotic stresses. OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5 -RNAi transgenic plants. To further investigate the biological functions of OsbZIP53 , we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants. Comprehensive analysis of phenotypical, physiological, and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants, but also regulated the immune response through the salicylic acid pathway. Specifically, disrupting OsbZIP53 increased H 2 O 2 accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs (Osrboh genes) and weakened H 2 O 2 degradation by directly targeting OsMYBS1. In addition, the growth of osbzip53 mutants was seriously impaired, while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type, suggesting that OsbZIP53 has a balancing effect on rice immune response and growth. [ABSTRACT FROM AUTHOR]

Our previous research showed that a powdery mildew resistant barley line (MvHV07-17) maintains its resistance to Blumeria hordei (Bh) even if plants are exposed to a long-term high temperature of 35°C for 120 h before Bh inoculation, whereas such high temperature pretreatment further increases susceptibility to infection in the susceptible barley line MvHV118-17. In the present study, we extended this approach using short-term high-temperature water treatment (49°C for 30 s) to determine how it affects powdery mildew resistance in these barley lines. We found that this short-term heat shock (HS) impaired plant defense responses, as reflected by development of Bh colonies and visible necrotic spots on leaves of MvHV07-17, which does not develop visible symptoms upon Bh inoculation under optimal growth conditions. In contrast, both HS and long-term heat stress enhanced susceptibility to Bh in MvHV118-17 plants. These results were supported by the measurement of Bh biomass using a qPCR method. Furthermore, microscopic examinations showed that HS elevated the rate of successful Bh penetration events and the spread of cell death in the surrounding mesophyll area and allowed for colony formation and sporulation in resistant barley, whereas early and effective plant defense responses, such as papilla formation and single-cell epidermal hypersensitive response, were significantly reduced. Furthermore, we found that the accumulation of hydrogen peroxide in both resistant and susceptible barley was correlated with susceptibility induced by HS and long-term heat-stress. This study may contribute to a better understanding of plant defense responses to Bh in barley exposed to heat. [ABSTRACT FROM AUTHOR]

Abstract Accurate quantification of hypersensitive response (HR) programmed cell death is imperative for understanding plant defense mechanisms and developing disease‐resistant crop varieties. Here, a phenotyping platform for rapid, continuous‐time, and quantitative assessment of HR is demonstrated: Parallel Automated Spectroscopy Tool for Electrolyte Leakage (PASTEL). Compared to traditional HR assays, PASTEL significantly improves temporal resolution and has high sensitivity, facilitating detection of microscopic levels of cell death. Validation is performed by transiently expressing the effector protein AVRblb2 in transgenic Nicotiana benthamiana (expressing the corresponding resistance protein Rpi‐blb2) to reliably induce HR. Detection of cell death is achieved at microscopic intensities, where leaf tissue appears healthy to the naked eye one week after infiltration. PASTEL produces large amounts of frequency domain impedance data captured continuously. This data is used to develop supervised machine‐learning (ML) models for classification of HR. Input data (inclusive of the entire tested concentration range) is classified as HR‐positive or negative with 84.1% mean accuracy (F1 score = 0.75) at 1 h and with 87.8% mean accuracy (F1 score = 0.81) at 22 h. With PASTEL and the ML models produced in this work, it is possible to phenotype disease resistance in plants in hours instead of days to weeks.

Abstract Sorghum relies on its immune system to defend against various pathogens, including Pseudomonas syringae syringae van Hall (Pss van Hall). However, the sorghum immune system is largely unknown. Reports on pathogenic effectors have provided valuable insights into the plant immune system; thus, we aimed to identify Pss van Hall effectors that can regulate the sorghum defense response in this study. Here, we first established the sorghum-Pss van Hall pathosystem and found that type III effectors played critical roles in the virulence of Pss van Hall to sorghum. To predict its effectors, the whole genome of Pss van Hall was sequenced, and 18 effector-coding genes were identified. Among them, five effectors belong to the core effectors of Pseudomonas syringae pathovars, and two may be monocot pathogen-specific effectors. Pss van Hall triggered the hypersensitive response (HR) in Nicotiana benthamiana. We found that the effectors of Pss van Hall can be divided into cell death inducers and immune repressors by examining their functions in HR induction and repression of PTI marker gene, ROS production, and pathogen growth. Finally, the roles of core effectors HopAJ2 and HopAN1, and specific effector HopAX1 were further confirmed in the sorghum-Pss van Hall pathosystem. Importantly, the functions of HopAN1 and HopAX1 in regulating plant immunity were reported for the first time. We believe that the identification of these effectors will facilitate the continued exploration of the sorghum immune system.

Abstract Background Purple curl leaf disease brings a significant threat to the development of agave industry, the underlying mechanism of disease-resistant Agave sisalana. hybrid 11648 (A. H11648R) is still unknown. Results To excavate the crucial disease-resistant genes against purple curl leaf disease, we performed an RNA-seq analysis for A.H11648R and A.H11648 during different stages of purple curl leaf disease. The DEGs (differentially expressed genes) were mainly enriched in linolenic acid metabolism, starch and sucrose mechanism, phenylpropanoid biosynthesis, hypersensitive response (HR) and systemic acquired resistance. Further analysis suggested that eight candidate genes (4’OMT2, ACLY, NCS1, GTE10, SMO2, FLS2, SQE1 and RCOM) identified by WGCNA (weighted gene co-expression network analysis) may mediate the resistance to agave purple curl disease by participating the biosynthesis of benzylisoquinoline alkaloids, steroid, sterols and flavonoids, and the regulation of plant innate immunity and systemic acquired resistance. After qPCR verification, we found that AsRCOM, coding a glycosyltransferase and relevant to the regulation of plant innate immunity and systemic acquired resistance, may be the most critical disease-resistant gene. Finally, the overexpression of AsRCOM gene in agave could significantly enhance the resistance to purple curl disease with abundant reactive oxygen species (ROS) accumulations. Conclusions Integrative RNA-seq analysis found that HR may be an important pathway affecting the resistance to purple curl leaf disease in agave, and identified glycosyltransferase AsRCOM as the crucial gene that could significantly enhance the resistance to purple curl leaf disease in agave, with obvious ROS accumulations.

Whitefly-transmitted begomoviruses infect and damage a wide range of food, feed, and fiber crops worldwide. Some of these viruses are associated with betasatellite molecules that are known to enhance viral pathogenesis. In this study, we investigated the function of a novel βV1 protein encoded by radish leaf curl betasatellite (RaLCB) by overexpressing the protein using potato virus X (PVX)-based virus vector in Nicotiana benthamiana. βV1 protein induced lesions on leaves, suggestive of hypersensitive response (HR), indicating cell death. The HR reaction induced by βV1 protein was accompanied by an increased accumulation of reactive oxygen species (ROS), free radicals, and HR-related transcripts. Subcellular localization through confocal microscopy revealed that βV1 protein localizes to the cellular periphery. βV1 was also found to interact with replication enhancer protein (AC3) of helper virus in the nucleus. The current findings suggest that βV1 functions as a protein elicitor and a pathogenicity determinant.

Movement proteins (MPs) encoded by plant viruses are essential for cell-to-cell transport of viral genomes through plasmodesmata. The genome of hibiscus green spot virus contains a module of two MP genes termed ‘binary movement block’ (BMB), encoding the proteins BMB1 and BMB2. Here, BMB1 is shown to induce a defense response in Nicotiana benthamiana plants that inhibits BMB-dependent virus transport. This response is characterized by the accumulation of reactive oxygen species, callose deposition in the cell wall, and upregulation of 9-LOX expression. However, the BMB1-induced response is inhibited by coexpression with BMB2. Furthermore, BMB1 is found to localize to subnuclear structures, in particular to Cajal bodies, in addition to the cytoplasm. As shown in experiments with a BMB1 mutant, the localization of BMB1 to nuclear substructures enhances BMB-dependent virus transport. Thus, the virus transport mediated by BMB proteins is modulated by (i) a BMB1-induced defense response that inhibits transport, (ii) suppression of the BMB1-induced response by BMB2, and (iii) the nuclear localization of BMB1 that promotes virus transport. Collectively, the data presented demonstrate multiple levels of interactions between viral pathogens and their plant hosts during virus cell-to-cell transport.

Sorghum relies on its immune system to defend against various pathogens, including Pseudomonas syringae syringae van Hall (Pss van Hall). However, the sorghum immune system is largely unknown. Reports on pathogenic effectors have provided valuable insights into the plant immune system; thus, we aimed to identify Pss van Hall effectors that can regulate the sorghum defense response in this study. Here, we first established the sorghum-Pss van Hall pathosystem and found that type III effectors played critical roles in the virulence of Pss van Hall to sorghum. To predict its effectors, the whole genome of Pss van Hall was sequenced, and 18 effector-coding genes were identified. Among them, five effectors belong to the core effectors of Pseudomonas syringae pathovars, and two may be monocot pathogen-specific effectors. Pss van Hall triggered the hypersensitive response (HR) in Nicotiana benthamiana. We found that the effectors of Pss van Hall can be divided into cell death inducers and immune repressors by examining their functions in HR induction and repression of PTI marker gene, ROS production, and pathogen growth. Finally, the roles of core effectors HopAJ2 and HopAN1, and specific effector HopAX1 were further confirmed in the sorghum-Pss van Hall pathosystem. Importantly, the functions of HopAN1 and HopAX1 in regulating plant immunity were reported for the first time. We believe that the identification of these effectors will facilitate the continued exploration of the sorghum immune system. [ABSTRACT FROM AUTHOR]

Background: Purple curl leaf disease brings a significant threat to the development of agave industry, the underlying mechanism of disease-resistant Agave sisalana. hybrid 11648 (A. H11648R) is still unknown. Results: To excavate the crucial disease-resistant genes against purple curl leaf disease, we performed an RNA-seq analysis for A.H11648R and A.H11648 during different stages of purple curl leaf disease. The DEGs (differentially expressed genes) were mainly enriched in linolenic acid metabolism, starch and sucrose mechanism, phenylpropanoid biosynthesis, hypersensitive response (HR) and systemic acquired resistance. Further analysis suggested that eight candidate genes (4'OMT2, ACLY, NCS1, GTE10, SMO2, FLS2, SQE1 and RCOM) identified by WGCNA (weighted gene co-expression network analysis) may mediate the resistance to agave purple curl disease by participating the biosynthesis of benzylisoquinoline alkaloids, steroid, sterols and flavonoids, and the regulation of plant innate immunity and systemic acquired resistance. After qPCR verification, we found that AsRCOM, coding a glycosyltransferase and relevant to the regulation of plant innate immunity and systemic acquired resistance, may be the most critical disease-resistant gene. Finally, the overexpression of AsRCOM gene in agave could significantly enhance the resistance to purple curl disease with abundant reactive oxygen species (ROS) accumulations. Conclusions: Integrative RNA-seq analysis found that HR may be an important pathway affecting the resistance to purple curl leaf disease in agave, and identified glycosyltransferase AsRCOM as the crucial gene that could significantly enhance the resistance to purple curl leaf disease in agave, with obvious ROS accumulations. [ABSTRACT FROM AUTHOR]

Wheat leaf rust (Puccinia triticina) is one of the most significant fungal diseases of wheat, causing substantial yield losses worldwide. Infestation is currently being reduced by fungicide treatments and mostly vertical resistance. However, these measures often break down when the fungal virulence pattern changes, resulting in a breakdown of vertical resistances. In contrast, the prehaustorial resistance (phr) that occurs in the einkorn–wheat leaf rust interaction is race-independent, characterized by an early defense response of plants during the prehaustorial phase of infestation. Einkorn (Triticum monococcum) is closely related to Triticum urartu as a progenitor of wheat and generally shows a high level of resistance against leaf rust of wheat. Hence, einkorn can serve as a valuable source to improve the level of resistance to the pathogen in future wheat lines. In particular, einkorn accession PI272560 is known to exhibit a hypersensitive prehaustorial effector triggered immune reaction, preventing the infection of P. triticina. Remarkably, this effector-triggered immune reaction turned out to be atypical as it is non-race-specific (horizontal). To genetically dissect the prehaustorial resistance (phr) in PI272560, a biparental F2 population of 182 plants was established after crossing PI272560 with the susceptible T. boeoticum accession 36554. Three genetic maps comprising 2,465 DArT-seq markers were constructed, and a major QTL was detected on chromosome 5A. To locate underlying candidate genes, marker sequences flanking the respective QTL were aligned to the T. urartu reference genome and transcriptome data available from the parental accessions were used. Within the QTL interval of approximately 16.13 million base pairs, the expression of genes under inoculated and non- inoculated conditions was analyzed via a massive analysis of cDNA (MACE). Remarkably, a single gene located 3.4 Mbp from the peak marker within the major QTL was upregulated (20- to 95-fold) after the inoculation in the resistant accession in comparison to the susceptible T. boeoticum accession. This gene belongs to a berberine bridge enzyme-like protein that is suspected to interact on the plant surface with glycoside hydrolases (GH) secreted by the fungus and to induce a hypersensitive defense reaction in the plant after fungal infections. [ABSTRACT FROM AUTHOR]

Brown rot, caused by the Monilinia spp., is the disease that causes the greatest losses in stone fruit worldwide. Currently, M. fructicola has become the dominant species in the main peach production area in Spain. The fruit cuticle is the first barrier of protection against external aggressions and may have a key role in the susceptibility to brown rot. However, information on the role of skin fruit on the resistance to brown rot in peach is scarce. Previous genetic analyses in peach have demonstrated that brown rot resistance is a complex and quantitative trait in which different fruit parts and resistance mechanisms are involved. To search for genomic areas involved in the control of the cultivar susceptibility to brown rot and to elucidate the role of fruit skin against this infection, we have studied, for two consecutive seasons (2019 and 2020), the fruit susceptibility to M. fructicola, together with fruit cuticle thickness (CT) and density (CD), in a collection of 80 Spanish and 5 foreign peach cultivars from the National Peach Collection at CITA (Zaragoza, Spain). Brown rot incidence, lesion diameter, and severity index were calculated after 5 days of inoculation on non-wounded fruit. The peach collection has also been genotyped using the new peach SNP chip (9 + 9K). Genotypic and phenotypic data have been used to perform a genome- wide association analysis (GWAS). Phenotyping has shown a wide variability on the brown rot susceptibility within the Spanish germplasm as well as on CD and CT. The GWAS results have identified several significant SNPs associated with disease severity index (DSI), CD, and CT, five of which were considered as reliable SNP-trait associations. A wide protein network analysis, using 127 genes within the regions of the reliable SNPs and previously identified candidate genes (169) associated with Monilinia spp. resistance, highlighted several genes involved in classical hypersensitive response (HR), genes related to wax layers as ceramidases and lignin precursors catalyzers, and a possible role of autophagy during brown rot infection. This work adds relevant information on the complexity resistance mechanisms to brown rot infection in peach fruits and the genetics behind them. [ABSTRACT FROM AUTHOR]

Imbibing watermelon seeds in 1 mM sodium tetraborate (Na2B4O7) for 24 h systemically protected plants against foliar infection by Stagonosporopsis cucurbitacearum in detached leaves and under greenhouse conditions. The treatment resulted in both a reduction in the overall percentage of leaf infection as well as in the size of lesions. Studies of the mechanisms by which Na2B4O7 protected watermelon showed that there was no direct effect on the S. cucurbitacearum mycelium growth in vitro. On the other hand, plants raised from seeds primed with Na2B4O7 showed a higher frequency of fluorescent epidermal cells compared to the plants treated with water. This indicates that a higher number of cells expressed the hypersensitive response after Na2B4O7 priming. In addition, there was an increase in peroxidase activity and an enhanced accumulation of a 45 kDa acidic peroxidase isoform during the early stages of infection in plants treated with Na2B4O7 compared to plants treated with water and this was positively correlated to the reduction of leaf infection caused by the pathogen. These results indicate that Na2B4O7 is able to induce systemic resistance in watermelon against S. cucurbitacearum by activating the hypersensitive reaction at penetration sites, increasing peroxidase activity and altering the peroxidase isozyme profile. Although each individual response may only have had a minor effect, their combined effects had a reducing effect on the disease.

Wheat leaf rust (Puccinia triticina) is one of the most significant fungal diseases of wheat, causing substantial yield losses worldwide. Infestation is currently being reduced by fungicide treatments and mostly vertical resistance. However, these measures often break down when the fungal virulence pattern changes, resulting in a breakdown of vertical resistances. In contrast, the prehaustorial resistance (phr) that occurs in the einkorn–wheat leaf rust interaction is race-independent, characterized by an early defense response of plants during the prehaustorial phase of infestation. Einkorn (Triticum monococcum) is closely related to Triticum urartu as a progenitor of wheat and generally shows a high level of resistance against leaf rust of wheat. Hence, einkorn can serve as a valuable source to improve the level of resistance to the pathogen in future wheat lines. In particular, einkorn accession PI272560 is known to exhibit a hypersensitive prehaustorial effector triggered immune reaction, preventing the infection of P. triticina. Remarkably, this effector-triggered immune reaction turned out to be atypical as it is non-race-specific (horizontal). To genetically dissect the prehaustorial resistance (phr) in PI272560, a biparental F2 population of 182 plants was established after crossing PI272560 with the susceptible T. boeoticum accession 36554. Three genetic maps comprising 2,465 DArT-seq markers were constructed, and a major QTL was detected on chromosome 5A. To locate underlying candidate genes, marker sequences flanking the respective QTL were aligned to the T. urartu reference genome and transcriptome data available from the parental accessions were used. Within the QTL interval of approximately 16.13 million base pairs, the expression of genes under inoculated and non-inoculated conditions was analyzed via a massive analysis of cDNA (MACE). Remarkably, a single gene located 3.4 Mbp from the peak marker within the major QTL was upregulated (20- to 95-fold) after the inoculation in the resistant accession in comparison to the susceptible T. boeoticum accession. This gene belongs to a berberine bridge enzyme-like protein that is suspected to interact on the plant surface with glycoside hydrolases (GH) secreted by the fungus and to induce a hypersensitive defense reaction in the plant after fungal infections.

Brown rot, caused by the Monilinia spp., is the disease that causes the greatest losses in stone fruit worldwide. Currently, M. fructicola has become the dominant species in the main peach production area in Spain. The fruit cuticle is the first barrier of protection against external aggressions and may have a key role in the susceptibility to brown rot. However, information on the role of skin fruit on the resistance to brown rot in peach is scarce. Previous genetic analyses in peach have demonstrated that brown rot resistance is a complex and quantitative trait in which different fruit parts and resistance mechanisms are involved. To search for genomic areas involved in the control of the cultivar susceptibility to brown rot and to elucidate the role of fruit skin against this infection, we have studied, for two consecutive seasons (2019 and 2020), the fruit susceptibility to M. fructicola, together with fruit cuticle thickness (CT) and density (CD), in a collection of 80 Spanish and 5 foreign peach cultivars from the National Peach Collection at CITA (Zaragoza, Spain). Brown rot incidence, lesion diameter, and severity index were calculated after 5 days of inoculation on non-wounded fruit. The peach collection has also been genotyped using the new peach SNP chip (9 + 9K). Genotypic and phenotypic data have been used to perform a genome-wide association analysis (GWAS). Phenotyping has shown a wide variability on the brown rot susceptibility within the Spanish germplasm as well as on CD and CT. The GWAS results have identified several significant SNPs associated with disease severity index (DSI), CD, and CT, five of which were considered as reliable SNP-trait associations. A wide protein network analysis, using 127 genes within the regions of the reliable SNPs and previously identified candidate genes (169) associated with Monilinia spp. resistance, highlighted several genes involved in classical hypersensitive response (HR), genes related to wax layers as ceramidases and lignin precursors catalyzers, and a possible role of autophagy during brown rot infection. This work adds relevant information on the complexity resistance mechanisms to brown rot infection in peach fruits and the genetics behind them.

Virus-specific proteins, including coat proteins, movement proteins, replication proteins, and suppressors of RNA interference are capable of triggering the hypersensitive response (HR), which is a type of cell death in plants. The main cell death signaling pathway involves direct interaction of HR-inducing proteins with nucleotide-binding leucine-rich repeats (NLR) proteins encoded by plant resistance genes. Singleton NLR proteins act as both sensor and helper. In other cases, NLR proteins form an activation network leading to their oligomerization and formation of membrane-associated resistosomes, similar to metazoan inflammasomes and apoptosomes. In resistosomes, coiled-coil domains of NLR proteins form Ca2+ channels, while toll-like/interleukin-1 receptor-type (TIR) domains form oligomers that display NAD+ glycohydrolase (NADase) activity. This review is intended to highlight the current knowledge on plant innate antiviral defense signaling pathways in an attempt to define common features of antiviral resistance across the kingdoms of life. [ABSTRACT FROM AUTHOR]

Phospholipid signaling plays important roles in plant immune responses. Here, we focused on two phospholipase C3 (PLC3) orthologs in the Nicotiana benthamiana genome, NbPLC3-1 and NbPLC3-2. We generated NbPLC3-1 and NbPLC3-2 -double-silenced plants (NbPLC3s -silenced plants). In NbPLC3s -silenced plants challenged with Ralstonia solanacearum 8107, induction of hypersensitive response (HR)-related cell death and bacterial population reduction was accelerated, and the expression level of Nbhin1 , a HR marker gene, was enhanced. Furthermore, the expression levels of genes involved in salicylic acid and jasmonic acid signaling drastically increased, reactive oxygen species production was accelerated, and NbMEK2 -induced HR-related cell death was also enhanced. Accelerated HR-related cell death was also observed by bacterial pathogens Pseudomonas cichorii , P. syringae , bacterial AvrA, oomycete INF1, and TMGMV-CP with L1 in NbPLC3s -silenced plants. Although HR-related cell death was accelerated, the bacterial population was not reduced in double NbPLC3s and NbCoi1 -suppressed plants nor in NbPLC3s -silenced NahG plants. HR-related cell death acceleration and bacterial population reduction resulting from NbPLC3s- silencing were compromised by the concomitant suppression of either NbPLC3 s and NbrbohB (respiratory oxidase homolog B) or NbPLC3 s and NbMEK2 (mitogen activated protein kinase kinase 2). Thus, NbPLC3s may negatively regulate both HR-related cell death and disease resistance through MAP kinase- and reactive oxygen species-dependent signaling. Disease resistance was also regulated by NbPLC3s through jasmonic acid- and salicylic acid-dependent pathways. [ABSTRACT FROM AUTHOR]

The highly conserved angiosperm immune receptor HOPZ‐ACTIVATED RESISTANCE1 (ZAR1) recognises the activity of diverse pathogen effector proteins by monitoring the ZED1‐related kinase (ZRK) family. Understanding how ZAR1 achieves interaction specificity for ZRKs may allow for the expansion of the ZAR1‐kinase recognition repertoire to achieve novel pathogen recognition outside of model species. We took advantage of the natural diversity of Arabidopsis thaliana kinases to probe the ZAR1‐kinase interaction interface and found that A. thaliana ZAR1 (AtZAR1) can interact with most ZRKs, except ZRK7. We found evidence of alternative splicing of ZRK7, resulting in a protein that can interact with AtZAR1. Despite high sequence conservation of ZAR1, interspecific ZAR1‐ZRK pairings resulted in the autoactivation of cell death. We showed that ZAR1 interacts with a greater diversity of kinases than previously thought, while still possessing the capacity for specificity in kinase interactions. Finally, using AtZAR1‐ZRK interaction data, we rationally increased ZRK10 interaction strength with AtZAR1, demonstrating the feasibility of the rational design of a ZAR1‐interacting kinase. Overall, our findings advance our understanding of the rules governing ZAR1 interaction specificity, with promising future directions for expanding ZAR1 immunodiversity. SUMMARY STATEMENT: The angiosperm immune receptor ZAR1 displays specific interactions with diverse kinases, and rationally designed single amino acid substitutions in a kinase can affect interaction sensitivity. [ABSTRACT FROM AUTHOR]

Nucleotide-binding leucine-rich repeat receptors (NLRs) are the most abundant type of immune receptors in plants and can trigger a rapid cell-death (hypersensitive) response upon sensing pathogens. We previously cloned the wheat NLR Sr35, which encodes a coiled-coil (CC) NLR that confers resistance to the virulent wheat stem rust race Ug99. Here, we investigated Sr35 signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. Expression of Sr35 in N. benthamiana leaves triggered a mild cell-death response, which is enhanced in the autoactive mutant Sr35 D503V. The N-terminal tagging of Sr35 with green fluorescent protein (GFP) blocked the induction of cell death, whereas a C-terminal GFP tag did not. No domain truncations of Sr35 generated cell-death responses as strong as the wild type, but a truncation including the NB-ARC (nucleotide binding adaptor) shared by APAF-1, R proteins, and CED-4 domains in combination with the D503V autoactive mutation triggered cell death. In addition, coexpression of Sr35 with the matching pathogen effector protein AvrSr35 resulted in robust cell death and electrolyte leakage levels that were similar to autoactive Sr35 and significantly higher than Sr35 alone. Coexpression of Sr35-CC-NB-ARC and AvrSr35 did not induce cell death, confirming the importance of the leucine-rich repeat (LRR) domain for AvrSr35 recognition. These findings were confirmed through Agrobacterium-mediated transient expression in barley. Taken together, these results implicate the CC-NB-ARC domains of Sr35 in inducing cell death and the LRR domain in AvrSr35 recognition.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Abstract Increasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches’-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli-based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca. P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H2O2 accumulation in Nicotiana benthamiana, indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca. P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas.

Pistachio yield is annually reduced due to root-knot nematode infections. In order to evaluate its resistance to Meloidogyne javanica, three domestic pistachio rootstocks, namely, Badami, Ghazvini and Sarakhs, and a wild pistachio, Baneh (Pistacia atlantica subsp. mutica), were selected. Their response to the nematode infection was evaluated based on different plant and nematode indexes, 120 days post-inoculation (dpi). The penetration and development rate of nematode in roots of these four pistachio rootstocks were evaluated at different time points by acid fuchsin staining. Based on the measured indexes, Badami, Ghazvini, Sarakhs, and Baneh rootstocks ranked as susceptible, moderately resistant, moderately resistant, and resistant, respectively. The penetration rate of second-stage nematode juveniles (J2) into four rootstocks was discussed. The first “midstage” or swollen juveniles appeared at 4 dpi but to a lesser extent in Ghazvini, Sarakhs, and Baneh cultivars. The first females were seen in Badami at 21 dpi, in Ghazvini and Sarakhs at 35 dpi, and in Baneh at 45 dpi. Three types of defense responses were distinguished and characterized in the examined pistachio rootstocks: (i) a hypersensitive response (HR)-like reaction in the cortex in Ghazvini, Sarakhs, and Baneh root tips at 4 dpi and 6 dpi; (ii) an HR response, degrading J2 which induce giant cells in the vascular cylinder of all rootstocks, at 6 dpi and 10 dpi; and (iii) an HR response, degrading females and giant cells in the vascular cylinder of all rootstocks at 15 dpi onward. These observations open new fields of study in breeding programs of this crop.

Gibberellins are considered to play an important role in modulating plant–pathogen interactions. In order to find this, we studied the effect of GA4 on the interactions of Bipolaris sorokiniana (Scc.) (BIPOL) with Triticum aestivum. After inoculating the roots of hydroponically grown T. avestivum with BIPOL, the GA4 level shot up by 26%. The increased level of GA4 elicited a hypersensitive response (HR) in the T. aestivum seedlings. The elicitation in HR was confirmed through measurement of HR-inducing signaling compounds (i.e. c-di-GMP, cAMP, phosphatidic acid, pure and esterified oxo-phytodeinoic acid, and jasmonic acid). The concentration of HR-inducing signaling compounds in the treated T. aestivum seedlings was 31% higher compared to the control seedlings. Moreover, the induced HR response lowered the indole-3-acetic acid (IAA) level by 15% and the transzeatin (TZn) level by 22%. However, the application of 10 mM uniconazole through aerial spray inhibited GA4 biosynthesis in T. aestivum seedlings. Also, BIPOL colonized the roots of T. aestivum seedlings after uniconazole treatment and promoted seedling growth through increased relative growth rate (14%) and net assimilation rate (27%).

Sheath rot disease (SRD) is one of the most devastating diseases of Manchurian wild rice (MWR) (Zizania latifolia Griseb). Pilot experiments in our laboratory have shown that an MWR cultivar “Zhejiao NO.7”exhibits signs of SRD tolerance. To explore the responses of Zhejiao No. 7 to SRD infection, we used a combined transcriptome and metabolome analysis approach. A total of 136 differentially accumulated metabolites (DAMs, 114 up- and 22 down-accumulated in FA compared to CK) were detected. These up-accumulated metabolites were enriched in tryptophan metabolism, amino acid biosynthesis, flavonoids, and phytohormone signaling. Transcriptome sequencing results showed the differential expression of 11,280 genes (DEGs, 5,933 up-, and 5,347 downregulated in FA compared to CK). The genes expressed in tryptophan metabolism, amino acid biosynthesis, phytohormone biosynthesis and signaling, and reactive oxygen species homeostasis confirmed the metabolite results. In addition, genes related to the cell wall, carbohydrate metabolism, and plant-pathogen interaction (especially hypersensitive response) showed changes in expression in response to SRD infection. These results provide a basis for understanding the response mechanisms in MWR to FA attack that can be used for breeding SRD-tolerant MWR. [ABSTRACT FROM AUTHOR]

Our previous study identified that the RepA protein encoded by the oat dwarf virus (ODV) was responsible for inducing a strong hypersensitive response (HR) during the virus infection in non-host tobacco plants. However, little was known about the molecular mechanism of the RepA-elicited HR. Here, a RING-finger protein, which is described as NbRFP1 and is mainly located in the cytoplasm and nucleus in Nicotiana benthamiana cells, was confirmed to interact with RepA. In addition, the accumulation level of NbRFP1 in N. benthamiana leaves was enhanced by either ODV infection or by only RepA expression. The knockdown of NbRFP1 by a TRV-mediated virus-induced gene silencing markedly delayed the ODV or RepA-elicited HR. By contrast, the overexpression of NbRFP1 in N. benthamiana conferred enhanced resistance to ODV infection and promoted RepA-induced HR. Further mutation analysis showed that a RING-finger domain located in NbRFP1 plays important roles in modulating RepA-induced HR, as well as in mediating the interaction between NbRFP1 and RepA. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS)- and hypersensitive response (HR)- mediated cell death have long been known to play critical roles in plant immunity to pathogens. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) isa destructive wheatpathogen. Here, we report a quantitative analysis of the proportion of infected cells with local apoplastic ROS (apoROS) versus intracellular ROS (intraROS) accumulation in various wheat accessions that carry different disease resistance genes R genes) at a series of time points postinfection. The proportion of apoROS accumulation was 70 to 80% of the infected wheat cells detected in both compatible and incompatible host-pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses was detectedin 1 I to 15% of the infected wheat cells, mainly in wheat lines that carried nucleotide- binding leucine-rich repeat R genes (e. g., Pn13 F, Pm41, Tdfm60, MIIW72. and Pm69). The lines that carry unconventional R genes, Pm24 (Wheat Tandem Kinase 3) and pin42 (a recessive R gene), showed fewer intraROS responses. whereas 11 % of Pn124 line- infected epidermis cells still showed HR cell death. suggesting that different resistance pathways are activated there. Here. we also demonstrated that ROS could not act as a strong systemic signal for inducing high resistance to Bgt in wheat. although it induced the expression of pathogenesis-related genes. These results provide new insights into the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew. [ABSTRACT FROM AUTHOR]

Bacterial blight resistancegene Bi has received little attention since it was first described in 1950. A near-isogenic line (NIL) of Gossypium hirstitum cotton, AcBj, was generated in an otherwise bacterial-blight-susceptible Acala 44' background. The introgressed locus Bi in AcB.i conferred strong and broad-spectrum resistance to bacterial blight. Segregation patterns of test crosses under Oklahoma field conditions indicated that AcBs is likely homozygous for resistance at two loci with partial dominance gene action. controlled-environment conditions, two of the four copies of Bj were required for effective resistance. Contrary to expectations of gene-forgene theory, AcBj conferred high resistance toward isogenic strains of Xanthomonas citri subsp. malvaceamm carrying cloned avirulence genes ar,·134. avi·b7. avi -Bin, avi·BIOL and arrB] 02, respectively, and weaker resistance toward the strain carrying cloned avrb6. The hypothesis that each gene. in the absence of a polygenic complex, triggers sesquiterpenoid phytoalexin production was tested by measurement of cadalene and lacinilene phytoalexins during resistant responses in five NILs carrying different B genes, four other lines carrying multiple resistance genes. as well as susceptible Ac44E. Phytoalexin production was an obvious. but variable, response in all nine resistant lines. AcBj accumulated an order of magnitude more of all four phytoalexins than any of the other resistant NILs. Its total leve[s were comparable to those detected in OKI.2, a highly resistant line that possesses several B genes in a polygenic background. [ABSTRACT FROM AUTHOR]

Plum pox virus (PPV) infects Prunus trees across the globe, causing the serious Sharka disease. Breeding programs in the past 20 years have been successful, generating plum varieties hypersensitive to PPV that show resistance in the field. Recently, a single tree displaying typical PPV symptoms was detected in an orchard of resistant plums. The tree was eradicated, and infected material was propagated under controlled conditions to study the new PPV isolate. Performing overlapping PCR analysis, the viral sequence was reconstructed, cloned and tested for infectivity in different 'Jojo'-based resistant plums. The results confirmed that the isolate, named PPV-D 'Herrenberg' (PPVD-H), was able to infect all these varieties. Analyses of chimeras between PPVD-H and a PPV-D standard isolate (PPVD) revealed that the NIa region of PPD-H, carrying three amino acid changes, was enough to break the resistance of these plums. Experiments with single and double mutants showed that all changes were essential to preserve the escaping phenotype. Additionally, one of the changes at the VPg-NIapro junction suggested the involvement of controlled endopeptidase cleavage in the viral response. Transient expression experiments in Nicotiana benthamiana confirmed that NIa cleavage in PPVD-H was reduced, compared to PPVD, linking the observed behavior to an NIa cleavage modulation. [ABSTRACT FROM AUTHOR]

IntroductionThe type III effector RipAA of Ralstonia solanacearum GMI1000 plays a critical role in the incompatible interaction on Nicotiana benthamiana.MethodsThe RipAA was transiently expressed in N. benthamiana by Agrobacterium-mediated transformation. Chemical staining with trypan blue and DAB were conducted to examine the cell death and the accumulation of hydrogen peroxide (H2O2), respectively. The expression of the marker genes for salicylic acid (SA) and jasmonic acid (JA) signaling was evaluated by quantitative reverse transcription PCR (qRT-PCR). The proteins interacted with RipAA was identified from N. benthamiana by yeast two-hybrid and pull-down assays. A TRV-mediated gene silencing was used to assess the role of host gene in response to RipAA expression and R. solanacearum infection.Results and discussionRipAA induced the accumulation of hydrogen peroxide (H2O2) and genome DNA degradation in N. benthamiana, which were accompanied by a hypersensitive reaction. Simultaneously, the marker genes for salicylic acid (SA) signaling were induced and those for jasmonic acid (JA) signaling were reduced. N. benthamiana chloroplastic AtpB, the ATPase β subunit, was identified as an interactor with RipAA. The silencing of atpB in N. benthamiana resulted in the inability of RipAA to induce a hypersensitive response, a compatible interaction with GMI1000, and an enhanced sensitivity to bacterial wilt. Our data support the concept that RipAA determines host-range specificity by targeting the host chloroplastic AtpB.

Increasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches'-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli-based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca. P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H2O2 accumulation in Nicotiana benthamiana, indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca. P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas. [ABSTRACT FROM AUTHOR]

Downy mildew, caused by Peronospora variabilis, is the most important quinoa disease worldwide. However, little is known about the resistance mechanisms acting against this disease. The study goals were to identify quinoa accessions showing resistance to P. variabilis under Spanish field conditions and to characterize the resistance mechanism involved. Towards these objectives, a total amount of 229 accessions of Chenopodium quinoa and one accession of each of the species Chenopodiun berlandieri subs. nutillae, Chenopodium ugandae, and Chenopodium opulifolium were screened for resistance to P. variabilis under field conditions in Córdoba, Spain, during two seasons. The response to P. variabilis in the accessions showed a continuous distribution ranging from complete resistance to high susceptibility. Fifteen resistant and one susceptible accessions were selected for further histological studies. Histological results showed that resistance to downy mildew in quinoa acts mainly at the stage of colony establishment. In resistant accessions, no colonies were formed or success in colony establishment was significantly reduced compared with the susceptible control. Hypersensitive response was associated with colony abortion in a number of the resistant accessions. This work is the first proof of hypersensitive reaction occurrence in quinoa as a response to P. variabilis. [ABSTRACT FROM AUTHOR]

Lentil rust is a major disease worldwide caused by Uromyces viciae-fabae. In this study, we screened a large germplasm collection of cultivated lentils (Lens culinaris ssp. culinaris) and its wild relatives, both in adult plants in the field with a local rust isolate during 2 seasons and in seedlings under controlled conditions with four fungal isolates of worldwide origin. The main results from our study were the following: (1) a significant number of accessions with resistance based on hypersensitive reaction (reduced Infection Type (IT)) were identified in cultivated lentil and in L. ervoides, L. nigricans and L.c. orientalis. The IT scores showed a clear isolate-specific response suggesting race-specificity, so each fungal isolate might be considered a different race. Resistance was identified against all isolates what might be the basis to develop a standard differential set that should be a priority for rust definition and monitoring. (2) Interestingly, although at lower frequency than in L. ervoides and L. nigricans, the hypersensitive response was also observed within cultivated lentil, with accession 1561 (L.c. culinaris) displaying resistance to the four isolates making this accession a valuable ready-to-use resource for lentil resistance breeding. Resistance to all other rust isolates was also available within L.c. culinaris in an isolate-specific manner. Accession 1308 (L. ervoides) showed resistance against all isolates tested, as well as a reduced number of accessions belonging to other wild Lens species. (3) In addition, our screenings allowed the identification of several accessions with partial resistance (reduced Disease Severity (DS) despite high IT). Adult Plant Resistance resulting in reduced severity in adult plants in the field, despite high susceptibility in seedlings, was more frequently identified in L.c. culinaris, but also in L. nigricans and L.c. orientalis. [ABSTRACT FROM AUTHOR]

Horticultural crops suffer from bacterial, fungal, and oomycete pathogens. Effectors are one of the main weapons deployed by those pathogens, especially in the early stages of infection. Pathogens secrete effectors with diverse functions to avoid recognition by plants, inhibit or manipulate plant immunity, and induce programmed cell death. Most identified effectors are proteinaceous, such as the well-studied type-III secretion system effectors (T3SEs) in bacteria, RXLR and CRN (crinkling and necrosis) motif effectors in oomycetes, and LysM (lysin motifs) domain effectors in fungi. In addition, some non-proteinaceous effectors such as toxins and sRNA also play crucial roles in infection. To cope with effectors, plants have evolved specific mechanisms to recognize them and activate effector-triggered immunity (ETI). This review summarizes the functions and mechanisms of action of typical proteinaceous and non-proteinaceous effectors secreted by important horticultural crop pathogens. The defense responses of plant hosts are also briefly introduced. Moreover, potential application of effector biology in disease management and the breeding of resistant varieties is discussed. [ABSTRACT FROM AUTHOR]

Due to the restricted genetic diversity among current asparagus cultivars, wild relatives are of particular interest as a source of resistance. In this study, seventeen cultivars and wild relatives were tested for their resistance to Fusarium oxysporum in the seedling test. Two isolates differing in virulence (single spore lines with high and low virulence) were used for testing. Most of the tested genotypes showed high infestation, whereby Asparagus aethiopicus caused no browning of the roots or growth depression. This wild relative was evaluated for its defence response after infection with F. oxysporum. For comparison, the wild relative Asparagus densiflorus and Asparagus officinalis cv. Thielim were tested. Like A. densiflorus, A. aethiopicus showed accumulation of hydrogen peroxide as a defence mechanism, while Asparagus officinalis cv. Thielim showed no storage of hydrogen compounds. [ABSTRACT FROM AUTHOR]

Summary: The oomycete Albugo candida causes white blister rust, an important disease of Brassica crops. Distinct races of A. candida are defined by their capacity to infect different host plant species. Each A. candida race encodes secreted proteins with a CX2CX5G ('CCG') motif that are polymorphic and show presence/absence variation, and are therefore candidate effectors.The White Rust Resistance 4 (WRR4) locus in Arabidopsis thaliana accession Col‐0 contains three genes that encode intracellular nucleotide‐binding domain leucine‐rich repeat immune receptors. The Col‐0 alleles of WRR4A and WRR4B confer resistance to multiple A. candida races, although both WRR4A and WRR4B can be overcome by the Col‐0‐virulent race 4 isolate AcEx1.Comparison of CCG candidate effectors in avirulent and virulent races, and transient co‐expression of CCG effectors from four A. candida races in Nicotiana sp. or A. thaliana, revealed CCG effectors that trigger WRR4A‐ or WRR4B‐dependent hypersensitive responses.We found eight WRR4A‐recognised CCGs and four WRR4B‐recognised CCGs, the first recognised proteins from A. candida for which the cognate immune receptors in A. thaliana are known. This multiple recognition capacity potentially explains the broad‐spectrum resistance to several A. candida races conferred by WRR4 paralogues. We further show that of five tested CCGs, three confer enhanced disease susceptibility when expressed in planta, consistent with A. candida CCG proteins being effectors. [ABSTRACT FROM AUTHOR]

Keywords: Effector; encasement; GEF; hypersensitive response; MVB; powdery mildew EN Effector encasement GEF hypersensitive response MVB powdery mildew 12 15 4 12/27/22 20230101 NES 230101 Membrane trafficking is key for different aspects of plant immunity such as the formation of papillae and haustorial encasements. The transport, tethering, and docking of target membranes is regulated by Rab GTPase proteins, which interact with membrane protein complexes to specify the tethering of membrane compartments ([6]). It is somewhat unsurprising that a I Bh i effector, such as CSEP0162, targets MON1 in an attempt to block immune responses, since membrane trafficking has a critical role in plant immunity during both pre- and post-invasion defences. As CSEP0162 may target MON1 to prevent MVB vesicle fusion to the plasma membrane, thereby preventing the HR, it will be interesting to test if the HR mediated by other CNLs (other than I Mla3 i ) is dependent on MON1. [Extracted from the article]

In this study, we identified a new citrus vein enation virus (CVEV) isolate (named CVEV-DT1) through sRNA high-throughput sequencing and traditional sequencing. Phylogenetic analysis based on whole genome sequences of all known CVEV isolates revealed that CVEV-DT1 was in an evolutionary branch with other isolates from China. Molecular variation analysis showed that the single nucleotide variability along CVEV full-length sequences was less than 8%, with more transitions (60.55%) than transversions (39.43%), indicating a genetically homogeneous CVEV population. In addition, non-synonymous nucleotide mutations mainly occurred in ORF1 and ORF2. Based on disorder analysis of all encoded ORF by CVEV-DT1, we identified that the CVEV-DT1 coat protein (CP) formed spherical granules, mainly in the cell nucleus and partly throughout the cytoplasm, with liquid properties through subcellular localization and photobleaching assay. Furthermore, we also confirmed that the CVEV P0 protein has weak post-transcriptional RNA-silencing suppressor activity and could elicit a strong hypersensitive response (HR) in tobacco plants. Collectively, to the best of our knowledge, our study was the first to profile the genomic variation in all the reported CVEV isolates and reveal the functions of CVEV-DT1-encoded proteins. [ABSTRACT FROM AUTHOR]