Search

Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM-like structures as anti-cancer vaccines in Nicotiana tabacum. High-mannose glycan structures were generated by adding a C-terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM-Fc and EpCAM-FcK proteins in transgenic plants via Agrobacterium-mediated transformation. Cross-fertilization of EpCAM-Fc (FcK) transgenic plants with Joining chain (J-chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM-IgA Fc (EpCAM-A) and IgM-like (EpCAM-M) proteins. Immunoblotting, SDS-PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti-EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti-EpCAM IgGs. Flow cytometry indicated that the EpCAM-Fc fusion proteins significantly activated CD8 + cytotoxic T cells, CD4 + helper T cells and B cells, particularly with EpCAM-FcK P and EpCAM-Fc P (FcK P ) × J P (JK P ). The induced anti-EpCAM IgGs captured human prostate cancer PC-3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down-regulated proliferation markers (PCNA, Ki-67) and epithelial-mesenchymal transition markers (Vimentin); and up-regulated E-cadherin. These findings suggest that N. tabacum can produce effective vaccine candidates to induce anti-cancer immune responses., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Salivary proteins secreted by phytophagous insects play pivotal roles in plant-insect interactions. A salivary protein RpSP27, from the stinkbug Riptortus pedestris, a devastating pest on soybean, was selected for studying due to its ability to induce cell death and activate immune responses in plants. RpSP27 localized to the endoplasmic reticulum and triggered reactive oxygen species burst. Virus-induced gene silencing assays showed RAR1 plays an essential role in RpSP27-induced cell death in Nicotiana benthamiana. Expression analyses revealed that RpSP27 is predominantly expressed in R. pedestris salivary glands. RNA interference-mediated silencing of RpSP27 in R. pedestris significantly reduced insect survival rates and altered feeding behavior by decreasing the formation of salivary sheaths on soybeans and reducing probing and feeding duration. Furthermore, the silencing of RpSP27 in R. pedestris mitigated the staygreen syndrome in soybeans, characterized by delayed senescence and pod abnormalities. This study elucidated the role of RpSP27 in the interaction between R. pedestris and soybean, presenting a potential target for pest management strategies to protect soybean crops from the detrimental effects of R. pedestris feeding., (© 2024 John Wiley & Sons Ltd.)

The protein kinase A/protein kinase G/protein kinase C-family (AGC kinase family) of eukaryotes is involved in regulating numerous biological processes. The 3-phosphoinositide- dependent protein kinase 1 (PDK1), is a conserved serine/threonine kinase in eukaryotes. To understand the roles of PDK1 homologous genes in cell death and immunity in tetraploid Nicotiana tabacum , the previuosly generated transgenic CRISPR/Cas9 lines, in which 5-7 alleles of the 4 homologous PDK1 genes ( NtPDK1a / 1b / 1c / 1d homologs) simultaneously knocked out, were used in this study. Our results showed that the hypersensitive response (HR) triggered by transient overexpression of active Pto (Pto Y207D ) or soybean Gm MEKK1 was significantly delayed, whereas the resistance to Pseudomonas syrangae pv. tomato DC3000 ( Pst DC3000) and tobacco mosaic virus (TMV) was significantly elevated in these partial knockout lines. The elevated resistance to Pst DC3000 and TMV was correlated with the elevated activation of Nt MPK6, Nt MPK3, and Nt MPK4. Taken together, our results indicated that NtPDK 1s play a positive role in cell death but a positive role in disease resistance, likely through negative regulation of the MAPK signaling cascade.

Proteins with Toll/interleukin-1 receptor (TIR) domains are widely distributed in both prokaryotes and eukaryotes, serving as essential components of immune signaling. Although monocots lack the major TIR nucleotide-binding leucine-rich repeat-type (TNL) immune receptors, they possess a small number of TIR-only proteins, the function of which remains largely unknown. In the monocot maize (Zea mays), there are 3 conserved TIR-only genes in the reference genome, namely ZmTIR1 to ZmTIR3. A genome-wide scan for TIR genes and comparative analysis revealed that these genes exhibit low sequence diversity and do not show copy number variation among 26 diverse inbred lines. ZmTIR1 and ZmTIR3, but not ZmTIR2, specifically trigger cell death and defense gene expression when overexpressed in Nicotiana benthamiana leaves. These responses depend on the critical glutamic acid and cysteine residues predicted to be essential for TIR-mediated NADase and 2',3'-cAMP/cGMP synthetase activity, respectively, as well as the key TIR downstream regulator Enhanced Disease Susceptibility 1 (EDS1). Overexpression of ZmTIR3 in N. benthamiana produces signaling molecules, including 2'cADPR, 2',3'-cAMP, and 2',3'-cGMP, a process that requires the enzymatic glutamic acid and cysteine residues of ZmTIR3. ZmTIR expression in maize is barely detectable under normal conditions but is substantially induced by different pathogens. Importantly, the maize Zmtir3 knockout mutant exhibits enhanced susceptibility to the fungal pathogen Cochliobolus heterostrophus, highlighting the role of ZmTIR3 in maize immunity. Overall, our results unveil the function of the maize ZmTIRs. We propose that the pathogen-inducible ZmTIRs play an important role in maize immunity, likely through their enzymatic activity and via EDS1-mediated signaling., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2025. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Tuta absoluta is a significant agricultural pest primarily affecting Solanaceae plants, resulting in substantial economic losses in agriculture. Insect saliva is an intermediary between insects and plants, playing a crucial role in modulating host adaptability and plant defense. This study analyzed the adaptive differences of T. absoluta on four plants using the two-sex life table method. Results indicated that the host adaptability of T. absoluta to tobacco is worse than its adaptability to the other three varieties of tomatoes. The salivary gland transcriptome analysis and signal peptide prediction revealed that Trypsin , B5 V51-1498 , and Ta74 were highly expressed in the salivary glands of T. absoluta subjected to tobacco treatment and exhibit the characteristics of secretory proteins, alongside significant feeding selection differences. Our findings elucidate the adaptive strategies of T. absoluta larvae on various Solanaceae plants and offer new insights into the salivary protein-mediated plant defense processes.

Salicylic acid (SA) plays important roles in plant local and systemic resistance. Isochorismate synthase 1 (ICS1) is a key enzyme in SA synthesis. Pathogens infection triggered the ICS1 expression and induced SA production. However, the molecular regulation mechanism of ICS1 against virus infection remains unclear. Here, we employed molecular genetics and physiobiochemical approaches to confirm a transcription factor NbNAC1 from Nicotiana benthamiana is a positive regulator of resistance against tobacco mosaic virus (TMV). The pathways NbNAC1 and NbICS1 can be triggered by TMV infection. Silencing NbNAC1 accelerated TMV-induced oxidative damage and increased reactive oxygen species (ROS) production. It also weakened both local and systemic resistance against TMV and decreased the expression of NbICS1, SA signaling gene NbNPR1, and SA defense-related genes. The effects of NbNAC1-silencing were restored by overexpression of NbICS1 or foliar SA applications. Overexpressing NbNAC1 prevented oxidative damage and reduced the production of ROS, enhanced plant resistance against viral pathogen, and activated NbICS1 expression, and SA downstream signaling and defense-related genes. NbNAC1 localized in nuclear and emerged the ability of transcriptional regulation. ChIP and EMSA results indicated that NbNAC1 directly binds to a fragment containing GAAATT motif of NbICS1 promoter. Luciferase reporter assays confirmed that NbNAC1 activates NbICS1 expression. Taken together, our results demonstrate that NbNAC1 plays a critical role in plant immunity through activation of SA production., (© 2025 Society for Experimental Biology and John Wiley & Sons Ltd.)

Citrus huanglongbing (HLB), caused by "Candidatus Liberibacter" spp., is one of the most disastrous citrus diseases worldwide. HLB-affected citrus fruits are significantly more acidic than healthy fruits. However, the molecular mechanism behind this phenomenon remains to be elucidated. Here, we report that HLB-affected fruits have higher levels of citric acid (CA) than healthy fruits. Moreover, Citrus PH4 (CitPH4), which encodes a MYB transcription factor that functions as a key regulator of CA accumulation, was upregulated in HLB-affected fruits relative to healthy fruits. Heterologous overexpression of CitPH4 in tobacco (Nicotiana tabacum) plants enhanced tolerance to HLB. Subsequently, overexpression and gene-editing experiments indicated that CitPH4 can affect the salicylic acid (SA) pathway, which directly binds to and activates the promoter of CsPBS3, a key gene of SA biosynthesis. HLB-affected fruits had higher SA levels than healthy fruits. Furthermore, application of SA activated CA biosynthesis and application of CA activated SA biosynthesis and signalling in citrus fruits and decreased "Candidatus Liberibacter asiaticus" (CLas) titres in infected leaves. This work suggests that CitPH4 is a key node between CA and SA, thus revealing crosstalk between defence responses and fruit quality in citrus., (© 2025 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The precise mechanisms by which plant viral proteases interact with and cleave host proteins, thereby participating in virus-host interactions, are not well understood. Potyviruses, the largest group of known plant-infecting RNA viruses, are known to rely on the nuclear inclusion protease a (NIa-Pro) for the processing of viral polyproteins. Here, we demonstrate that the proteolytic activity of NIa-Pro from potyvirus turnip mosaic virus (TuMV) is indispensable for inducing hypersensitive cell death in Nicotiana benthamiana. NIa-Pro targets and degrades the host DEAD-box protein 5 (DBP5) via a specific cleavage motif, which initiates host cell death. Both the silencing of DBP5 and the overexpression of NIa-Pro lead to an increased frequency of stop codon readthrough, which could be potentially harmful to the host, as it may result in the production of aberrant proteins. Unlike the NIa-Pro of most other potyviruses, the NIa-Pro of tobacco etch virus can also degrade DBP5 and trigger cell death, in both pepper and N. benthamiana. Furthermore, we discovered that the TuMV-encoded nuclear inclusion b can counteract NIa-Pro-induced cell death by co-opting DBP5. These findings unveil hitherto uncharacterized roles for plant virus proteases in cleaving host proteins and highlight the role of host DBP5 in modulating plant immunity., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Fusarium oxysporum, an important soilborne fungal pathogen that causes serious Fusarium wilt disease, secretes diverse effectors during the infection. In this study, we identified a novel secreted cysteine-rich protein, FolSCP1, which contains unknown protein functional domain. Here, we characterized FolSCP1 as a secreted virulence factor that promotes the pathogen infection of host plants by inhibiting diverse plant defence responses. FolSCP1 interacted with the pathogenesis-related 5 (PR-5) protein SlPR5, a positive regulator of tomato plant immunity against multiple tomato pathogens, and effectively attenuated the antifungal activity of the tomato PR-5 protein. FoSCP1, a homologue of FolSCP1, was secreted by a F. oxysporum isolate from infected tobacco and targeted the tobacco PR-5 protein NtPR5 to suppress plant defence for further infection. In summary, our study revealed a fungal virulence strategy in which F. oxysporum secrete effectors that interfere with plant immunity by binding to the PR-5 protein of the host plant and inhibiting its biological activity, thereby promoting fungal infection., (© 2024 John Wiley & Sons Ltd.)

Fruit is an essential part of the human diet, and postharvest fungal diseases are the major cause of fruit postharvest losses worldwide. Pathogen-associated molecular patterns (PAMPs) are important elicitors from microbes, and the recognition between microbial PAMPs and plant receptors leads to PAMP-triggered immunity. Here, we identified a PAMP, PdEIX, that is an important protein elicitor with plant immunity-inducing activity, from the citrus green mold pathogen Penicillium digitatum . PdEIX showed an apoplastic location similar to that of known PAMPs, and plant receptor-like protein NbEIX2, receptor-like kinase BAK1, and other signaling components of plant immunity were required for PdEIX-triggered plant cell death in the model plant Nicotiana benthamiana . Moreover, PdEIX triggered a series of immune responses in citrus fruit and enhanced the resistance of citrus and other fruit against fungal pathogens. Our results indicate that application of a microbial PAMP as the plant immunity inducer is an effective strategy for controlling postharvest fungal diseases of fruit.

Plants have developed various resistance mechanisms against herbivorous insects through prolonged coevolution. Plant defence responses can be triggered by specific compounds present in insect saliva. Apyrase, a known enzyme that catalyzes the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and inorganic phosphorus, has recently been identified in some herbivorous insects. However, whether insect salivary apyrase induces or inhibits plant responses remains poorly understood. In this study, we identified an apyrase-like protein in the salivary proteome of the fall armyworm, Spodoptera frugiperda, named Sfapyrase. Sfapyrase was primarily expressed in the salivary gland and secreted into plants during insect feeding. Transient expression of Sfapyrase in tobacco and maize enhanced plant resistance and resulted in decreased insect feeding. Knockdown of Sfapyrase through RNA interference led to increased growth and feeding of S. frugiperda. Furthermore, we showed that Sfapyrase activates the jasmonic acid signalling pathway and promotes the synthesis of secondary metabolites, especially benzoxazinoids, thereby enhancing resistance to S. frugiperda. In summary, our findings demonstrated that Sfapyrase acts as a salivary elicitor, inducing maize jasmonic acid defence responses and the production of insect-resistant benzoxazinoids. This study provides valuable insights into plant-insect interactions and offers potential targets for developing innovative insect pest management strategies., (© 2024 John Wiley & Sons Ltd.)

A predicted peanut R. solanacearum T3E RS_T3E_Hyp6 was identified as a definite T3E and renamed as RipBU. It is relative conserved in 31 R. solanacearum strains. Deletion of RipBU in R. solanacearum HA4-1 strain caused the attenuate pathogenicity in peanut, and complementarity of RipBU recovered the virulence of ΔRipBU mutant strain. Transient expression of RipBU decreased the level of chlorophyll, resulting in leaf chlorosis and suppressed flg22-triggered reactive oxygen species (ROS) burst and the expression of pattern-triggered immunity (PTI) marker genes in the leaves of Nicotiania benthamiana. Subcellular localization observation showed that RipBU localizes to chloroplasts in tobacco cells. RipBU significantly increased the jasmonic acid (JA) content and the expressions of JA-signaling marker genes in tobacco leaves, while significantly decreased the salicylic acid (SA) level and the expressions of SA-signaling marker genes. RipBU contained a putative lipase domain, and mutation of which abolished the ability of RipBU to induce tobacco leaf chlorosis and peanut wilt, while still localized to chloroplasts. Our study reveals the virulence function of RipBU that suppresses plant immunity by inhibiting PTI and SA signaling, and promoting JA signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cysteine-rich polycomb-like proteins (CPP) are crucial in regulating plant stress responses while the underlying functions of CPP involving plant- Ralstonia solanacaearum interaction remain unknown. Here, we showed the expression patterns of a potato CPP gene (StCPP3) under phytohormone treatments, biotic and abiotic stressed and its role in resistance against of R. solanacaearum infection by loss- and gain-of-function approaches. StCPP3 expression were up-regulated with methyl jasmonate (MeJA) and abscisic acid (ABA) while down-regulated under salicylic acid (SA), brassinosteroids (BR), high salt or low temperature treatment. Silencing the homolog gene (NbCPP3) in Nicotiana benthamiana enhanced resistance to R. solanacaearum. Over-expressing StCPP3 in Arabidopsis increased susceptibility and decreased activity of some defense-related enzymes, suggesting its role in suppressing hypersensitive cell death and reducing PR1 gene expression. In addition, we found that StCPP3 could interact with Type III secretion protein HrpB7 from R. solanacaearum. These results provide new insight into the mechanism of CPP's involvement in plant-pathogen interactions., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plants are equipped with multi-layered immune systems that recognize pathogen-derived elicitors to activate immunity. Verticillium dahliae is a soil-borne fungus that infects a broad range of plants and causes devastating wilt disease. The mechanisms underlying immune recognition between plants and V. dahliae remain elusive. Here, a V. dahliae secretory protein, elicitor of plant defense gene (VdEPD1), acts as an elicitor that triggers defense responses in both Nicotiana benthamiana and cotton plants is identified. Targeted gene deletion of VdEPD1 enhances V. dahliae virulence in plants. Expression of VdEPD1 triggers the accumulation of reactive oxygen species (ROS) and the activation of cell death in cotton plants. Gossypium barbadense EPD1-interacting receptor-like cytoplasmic kinase (GbEIR5A) and GbEIR5D interact with VdEPD1. Silencing of GbEIR5A/D significantly impairs VdEPD1-triggered cell death in cotton plants, indicating the contribution of GbEIR5A/D to VdEPD1-activated effector-triggered immunity (ETI). VdEPD1 stimulates the expression of GbEIR5A and GbEIR5D in cotton plants. Interestingly, cotton plants with silenced GbEIR5A/D genes exhibit compromised pathogen-associated molecular patterns (PAMPs)-triggered ROS accumulation, whereas overexpression of GbEIR5A or GbEIR5D enhances PAMP-induced ROS. These findings indicate that recognition of VdEPD1 potentiates GbEIRs to enhance cotton PAMP-triggered immunity (PTI), uncovering a cooperative interplay of PTI and ETI in cotton., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana. Agrobacterium-mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), N requirement gene 1 (NRG1) and SENESCENCE-ASSOCIATED GENE 101 (SAG101), indicating the activation of effector-triggered immunity (ETI) in N. benthamiana. Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1-1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll-interleukin-1 receptor (TIR) domain nucleotide-binding leucine-rich repeat receptor (TNL) called ROQ1 in N. benthamiana. Consistently, NlCSP11-induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA-degrading enzyme NahG. Through large-scale virus-induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana. Co-immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

Non-coding RNAs play crucial roles in plant responses to viral stresses. However, their molecular mechanisms in tea leaf spot responses remain unclear. In this study, using Camellia sinensis, we identified lncRNA81246 as a long non-coding RNA that localizes to both the nucleus and cytoplasm. It functions as a competitive endogenous RNA, thereby disrupting CsNAC1 (encoding NAC domain-containing protein 1) degradation mediated by miR164d. Silencing lncRNA81246 increased the resistance of tea plants to presistanceathogens, whereas transient lncRNA81246-overexpression plants showed decreased resistance to pathogens. Co-expression assays in Nicotiana benthamiana revealed that lncRNA81246 affects the miR164d-CsNAC1 regulatory module. Transient miR164d-overexpression and silencing assays demonstrated its positive regulation of tea plant resistance. Specifically, silencing its target, CsNAC1, enhanced disease resistance, whereas transient overexpression reduced plant resistance. Yeast one-hybrid, dual-luciferase, and RT-qPCR assay results suggested that CsNAC1 alters the expression of CsEXLB1, whereas AsODN and tobacco transient overexpression assays showed that CsEXLB1 negatively regulated tea plant resistance. Thus, our research demonstrated that lncRNA81246 acts as a mediator to interfere with the miR164d-CsNAC1 regulatory module involved in the disease resistance of tea plants., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Ralstonia solanacearum causes bacterial wilt, a devastating disease in solanaceous crops. The pathogenicity of R. solanacearum depends on its type III secretion system, which delivers a suite of type III effectors into plant cells. The disordered core effector RipAO is conserved across R. solanacearum species and affects plant immune responses when transiently expressed in Nicotiana benthamiana. Specifically, RipAO impairs pathogen-associated molecular pattern-triggered reactive oxygen species production, an essential plant defense mechanism. RipAO fused to yellow fluorescent protein initially localizes to filamentous structures, resembling the cytoskeleton, before forming large punctate aggregates around the nucleus. Consistent with these findings, tubulin alpha 6 (TUA6) and tubulin beta-1, building blocks of microtubules, were identified as putative targets of RipAO in immunoprecipitation and mass spectrometry analyses. In the presence of RipAO, TUA6-labeled microtubules fragmented into puncta, mimicking the effects of oryzalin, a microtubule polymerization inhibitor. These effects were corroborated in a N. benthamiana transgenic line constitutively expressing green fluorescent protein-labeled TUA6, where RipAO reduced microtubule density and stability at an accumulation level that did not induce aggregation. Moreover, oryzalin treatment further enhanced RipAO's impairment of reactive oxygen species production, suggesting that RipAO disrupts microtubule networks via its association with tubulins, leading to immune suppression. Further research into RipAO's interaction with the microtubule network will enhance our understanding of bacterial strategies to subvert plant immunity., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease affecting a great many crops including peanut. The pathogen damages plants via secreting type Ш effector proteins (T3Es) into hosts for pathogenicity. Here, we characterized RipAU was among the most toxic effectors as ΔRipAU completely lost its pathogenicity to peanuts. A serine residue of RipAU is the critical site for cell death. The RipAU targeted a subtilisin-like protease (AhSBT1.7) in peanut and both protein moved into nucleus. Heterotic expression of AhSBT1.7 in transgenic tobacco and Arabidopsis thaliana significantly improved the resistance to R. solanacearum. The enhanced resistance was linked with the upregulating ERF1 defense marker genes and decreasing pectin methylesterase (PME) activity like PME2&4 in cell wall pathways. The RipAU played toxic effect by repressing R-gene, defense hormone signaling, and AhSBTs metabolic pathways but increasing PMEs expressions. Furthermore, we discovered AhSBT1.7 interacted with AhPME4 and was colocalized at nucleus. The AhPME speeded plants susceptibility to pathogen via mediated cell wall degradation, which inhibited by AhSBT1.7 but upregulated by RipAU. Collectively, RipAU impaired AhSBT1.7 defense for pathogenicity by using PME-mediated cell wall degradation. This study reveals the mechanism of RipAU pathogenicity and AhSBT1.7 resistance, highlighting peanut immunity to bacterial wilt for future improvement., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae Kleb., is a destructive plant disease that instigates severe losses in many crops. Improving plant resistance to Verticillium wilt has been a challenge in most crops. In this study, a V. dahliae secreted protein VdSP8 was identified and shown to activate hyper-sensitive response (HR) and systemic acquired resistance (SAR) to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and Botrytis cinerea in tobacco plants. We identified a β-glucosidase named GhBGLU46 as a cotton plant target of VdSP8. VdSP8 interacts with GhBGLU46 both in vivo and in vitro and promotes the β-glucosidase activity of GhBGLU46. Silencing of GhBGLU46 reduced the expression of genes involved in lignin biosynthesis, such as GhCCR4, GhCCoAOMT2, GhCAD3 and GhCAD6, thus decreasing lignin deposition and increasing Verticillium wilt susceptibility. We have shown that GhBGLU46 is indispensable for the function of VdSP8 in plant resistance. These results suggest that plants have also evolved a strategy to exploit the invading effector protein VdSP8 to enhance plant resistance., (© 2024 John Wiley & Sons Ltd.)

The classic plant growth-promoting phytohormone cytokinin has been identified and established as a mediator of pathogen resistance in different plant species. However, the resistance effect of structurally different cytokinins appears to vary and may regulate diverse mechanisms to establish resistance. Hence, we comparatively analysed the impact of six different adenine- and phenylurea-type cytokinins on the well-established pathosystem Nicotiana tabacum-Pseudomonas syringae. The efficiency of resistance effects was evaluated based on impacts on the host plant defence response by scoring infection symptoms and the direct impact on the pathogen by assessment of proliferation in planta. To identify common and cytokinin-specific components involved in resistance effects, transcriptome profiling and targeted metabolomics were conducted in leaves treated with the different cytokinins. We observed clearly different potentials of the tested cytokinins in either suppressing infection symptoms or pathogen proliferation. Gene regulation and metabolite analyses revealed cytokinin-type specific impacts on defence components, such as salicylic acid and related signalling, expression of PR proteins, and regulation of specialised metabolism. Cytokinins also strongly affected plant cell physiological parameters, such as a remarkable decrease in amino acid pools. Hence, this study provides comparative information on the efficiency of diverse cytokinins in mediating resistance in one well-studied pathosystem and insights into the specific regulation of resistance effects mediated by different cytokinin molecules. This is particularly relevant for studies on the function of cytokinins or other phytohormones and compounds interacting with cytokinin activities in the context of pathogen infections and other stress scenarios, considering the diverse cytokinins present in plants., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Trans-acting small interfering RNAs (tasiRNAs) are 21-nt phased (phased siRNAs) resulting from successive DCL-catalyzed processing from the end of a double-stranded RNA substrate originating from the RDR of an AGO-catalyzed cleaved RNA at a micro RNA target site. Plant tasiRNAs have been synthesized to produce synthetic tasiRNAs (syn-tasiRNAs) targeting viral RNAs that confer viral resistance. In this study, we engineered syn-tasiRNAs to target potato virus Y (PVY) infection by replacing five native siRNAs of TAS1c with 210-bp fragments from the coat protein (CP) region of the PVY genome. The results showed that the transient expression of syn-tasiR-CPpvy2 in Nicotiana benthamiana (N. benthamiana) plants conferred antiviral resistance, supported by the absence of PVY infection symptoms and viral accumulation. This indicated that syn-tasiR-CPpvy2 successfully targeted and silenced the PVY CP gene, effectively inhibiting viral infection. syn-tasiR-CPpvy1 displayed attenuated symptoms and decreased viral accumulation in these plants However, severe symptoms of PVY infection and a similar amount of viral accumulation as the control were observed in plants expressing syn-tasiR-CPpvy3. syn-tasiR-CPpvy/pvx, which targets both PVY and potato virus X (PVX), was engineered using a single precursor. After the transient expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 in N. benthamiana, the plants were resistant to both PVY and PVX. These results suggested that engineered syn-tasiRNAs could not only specifically induce antiviral resistance against one target virus but could also be designed for multi-targeted silencing of different viruses, thereby preventing complex virus infection in plants.

Nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune receptors that activate innate immune responses upon sensing pathogen attack. However, the molecular mechanisms by which NLR proteins initiate downstream signal transduction pathways to counteract pathogen invasion remain poorly understood. In this study, we identified the wheat (Triticum aestivum) NLR protein Resistance Gene Analogs3 (TaRGA3), which was significantly upregulated during Puccinia striiformis f. sp. tritici (Pst) infection. TaRGA3 and its coiled-coil (CC) domain, localized to the cytoplasm and nucleus, can induce cell death in Nicotiana benthamiana. Virus-induced gene silencing and overexpression suggested that TaRGA3 contributed to wheat resistance to stripe rust by facilitating reactive oxygen species (ROS) accumulation. Yeast 2-hybrid, luciferase complementation imaging, and co-immunoprecipitation assays revealed that TaRGA3 interacted with wheat protein Ascorbate Peroxidase 6 (TaAPX6). Further analysis showed that TaAPX6 specifically targeted the CC domain of TaRGA3. The TaRGA3-TaAPX6 interplay led to reduced enzyme activity of TaAPX6. Notably, TaAPX6 negatively regulated wheat resistance to Pst by removing excessive ROS accompanying Pst-induced hypersensitive responses. Our findings reveal that TaRGA3 responding to Pst infection confers enhanced wheat resistance to stripe rust, possibly by suppressing TaAPX6-modulated ROS scavenging, and demonstrate that TaRGA3 can be used to engineer stripe rust resistance in wheat., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Ralstonia solanacearum causes lethal bacterial wilt diseases in numerous crops, resulting in considerable yield losses. Harnessing genetic resistance is desirable for safeguarding plants against phytopathogens. However, genetic resources resistant to bacterial wilt are limited in crops. RipE1, a conserved type Ⅲ effector with cysteine protease activity, is recognized in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana). Here, using a virus-induced gene silencing approach, we identified the gene encoding N. benthamiana homolog of Ptr1 (NbPtr1a), a coiled-coil nucleotide-binding leucine-rich repeat receptor (NLR) recognizing RipE1. Silencing or editing NbPtr1a completely abolished RipE1-induced cell death, indicating recognition of RipE1 by NbPtr1a. Genetic complementation confirmed this recognition, which is conserved across multiple solanaceous plants. Expression of RipE1 in planta or within pathogenic bacteria promoted pathogen colonization of Nbptr1a mutant plants, demonstrating its virulence function independent of NLR recognition. Silencing NbRIN4 enhanced RipE1-induced cell death, while expressing NbRIN4 inhibited it, suggesting that NbRIN4 is involved in recognition of NbPtr1a-RipE1. Furthermore, RipE1 associated with and cleaved NbRIN4, AtRIN4, and tomato (Solanum lycopersicum) SlRIN4 proteins through its cysteine protease activity. Silencing NbRIN4 in Nbptr1a mutants did not prevent RipE1 from promoting pathogen colonization, suggesting that NbRIN4 is not the primary target for RipE1-mediated virulence. Additionally, NbRIN4 suppressed self-association of the coiled-coil domain of NbPtr1a, which is critical for NbPtr1a-mediated cell death and resistance. Finally, we demonstrated that activation of NbPtr1a requires RipE1-mediated elimination of NbRIN4. Given the conserved nature of RipE1, Ptr1 holds great potential for protecting crops from diverse R. solanacearum strains and other distinct pathogens., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: NDR1/HIN1-like (NHL) genes play crucial roles in Psa resistance. Kiwifruit canker, caused by Pseudomonas syringae pv. Actinidiae (Psa) infection is one of the most serious diseases affecting the kiwifruit industry. However, the key NHL has not yet been identified in kiwifruit., Results: In this study, we conducted a genome-wide identification of NHL family in kiwifruit (Actinidia eriantha). A total of 33 AeNHLs were divided into five domain-conserved subfamilies, which were mainly assigned into phytohormones and defense responses. The expression of AeNHL genes was analyzed to identify key genes in response to Psa, and we found AeNHL17 was highly expressed upon Psa inoculation. Transgenic tobacco overexpressing AeNHL17 presented higher resistance to Psa than wild-type (WT) tobacco, implying a key role for AeNHL17 in Psa resistance. Finally, we carried out a stable genetic transformation of kiwifruit (A. chinensis), which is sensitive to Psa, and found that the overexpression of AeNHL17 increased resistance to infection. AeNHL17-silenced plants exhibited larger disease lesions than control plants., Conclusions: Our findings revealed the function of AaNHL17 in Psa resistance, providing new data regarding the functional analysis of the NHL gene family in kiwifruit., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Key Message: Transgenic tobacco plant expressed EpCAM-Fc fusion proteins to induce in vivo immune responses producing anti-EpCAM antibodies inhibiting human colorectal cancer cell invasion and migration. Plant is emerging as a promising alternative to produce valuable immunotherapeutic vaccines. In this study, we examined the in vivo anti-cancer efficacy of epidermal cell adhesion molecule (EpCAM)-Fc and EpCAM-FcK fusion proteins produced in transgenic plants as colorectal cancer vaccine candidates. Mice were injected with plant-derived EpCAM-Fc (EpCAM-Fc P ) and EpCAM-Fc P tagged with KDEL (ER retention signal) (EpCAM-FcK P ), using mammalian-derived EpCAM-Fc (EpCAM-Fc M ) as positive control. Total IgGs from the immunized mice were used to assess immune responses. ELISA tests revealed that IgGs from mice immunized with EpCAM-FcK P (EpCAM-FcK P IgG) exhibited the highest absorbance value for binding affinity to recombinant EpCAM-Fc M compared to IgGs from mice immunized with EpCAM-Fc P (EpCAM-Fc P IgG) and EpCAM-Fc M (EpCAM-Fc M IgG). Bio-layer interferometry revealed that EpCAM-FcK P IgG had a higher affinity value than EpCAM-Fc M IgG and EpCAM-Fc P IgG. Cell ELISA revealed that EpCAM-FcK P IgG exhibited the highest binding activity to EpCAM-positive cells SW480 and SW620 compared to EpCAM-Fc P IgG, EpCAM-Fc M IgG, and anti-EpCAM mAb. In the transwell invasion assay, EpCAM-FcK P IgG significantly decreased the numbers of invaded SW480 and SW620 cells compared to EpCAM-Fc P IgG, whereas EpCAM-Fc M IgG had similar numbers. In the wound healing assay, EpCAM-FcK P IgG showed higher migration inhibition compared to EpCAM-Fc P IgG in both cell types, with similar results to EpCAM-Fc M IgG in SW620 cells. These results confirm the applicability of plant systems to produce EpCAM-Fc vaccine candidates, inducing the production of anti-EpCAM IgGs against colorectal cancer cells., Competing Interests: Declarations. Conflict of interest: The authors have not disclosed any competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ningnanmycin has been proven to effectively inhibit infection by potato virus Y (PVY), although its underlying mechanisms remain unclear. In this study, we report a novel finding that ningnanmycin affects PVY replication. Two approaches were employed: studies using PVY movement-deficient mutants suggest that ningnanmycin, at a concentration of 500 μg/mL, inhibits PVY replication. Ningnanmycin interacts with the PVY-encoded coat protein (CP) with a binding constant of 1.34 μmol/L, and key amino acids Glu 168 and Thr 206 are involved in this interaction. Additionally, ningnanmycin induces the expression of antiviral response genes in Nicotiana benthamiana , including PRXIIB , PRXIIE , PUB4 , and PER42 . Furthermore, studies revealed that the overexpression of PUB4 in N. benthamiana confers resistance to PVY infection. These findings highlight the mechanisms by which ningnanmycin activates the PUB4 gene and suppresses CP assembly, thereby inhibiting PVY in N. benthamiana . This study represents an important step toward elucidating the molecular mechanism underlying the antiviral activity of ningnanmycin.

Plants must tactically balance immunity and growth when combating lethal pathogens like fungi. CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1), a conserved cell-surface co-receptor for the fungal elicitor chitin, enables plants to induce chitin-triggered immunity to counteract fungal invasion. Previously, we reported that bacterial infection can prime CERK1 through juxtamembrane (JM) phosphorylation to enhance fungal resistance, which only occurs in Arabidopsis (Arabidopsis thaliana) and its close relatives in Brassicaceae. Here, we aim to transfer the priming mechanism of Arabidopsis CERK1 (AtCERK1) to crop CERK1 via JM substitution. We revealed in protoplasts that the entire AtCERK1 JM variable region (AtJM) is essential for imparting the bacterial elicitor flg22-induced primed state to the Nicotiana benthamiana CERK1 (NbCERK1). The NbCERK1 chimera containing AtJM (NbCERK1AtJM) and similarly constructed rice (Oryza sativa) OsCERK1AtJM could undergo flg22-induced JM phosphorylation and confer enhanced antifungal immunity upon bacterial coinfection. Moreover, the NbCERK1AtJM+3D derivative with AtJM phosphomimetic mutations to mimic a constant primed state and similarly constructed OsCERK1AtJM+3D were sufficient to mediate strengthened chitin responses and fungal resistance in transgenic plants independent of bacterial infection. Importantly, no growth and reproduction defects were observed in these plants. Taken together, this study demonstrates that manipulating the primed state of a cell-surface immune receptor offers an effective approach to improve disease resistance in crops without compromising growth and yield and showcases how fundamental insights in plant biology can be translated into crop breeding applications., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The mirid bug (Riptortus pedestris), a major soybean pest, migrates into soybean fields during the pod filling stage and causes staygreen syndrome, which leads to substantial yield losses. The mechanism by which R. pedestris elicits soybean (Glycine max) defenses and counter-defenses remains largely unexplored. In this study, we characterized a protein family from R. pedestris, designated R.pedestris HAMP 1 (RPH1), and its putative paralogs (RPH1L1, 2, 3, 4, and 5), whose members exhibit dual roles in triggering and inhibiting plant immunity. RPH1 and RPH1L1 function as herbivore-associated molecular patterns (HAMPs), activating pattern-triggered immunity (PTI) in tobacco (Nicotiana benthamiana) and G. max. Furthermore, RPH1 stimulates jasmonic acid and ethylene biosynthesis in G. max, thereby enhancing its resistance to R. pedestris feeding. Additionally, RPH1 homologs are universally conserved across various herbivorous species, with many homologs also acting as HAMPs that trigger plant immunity. Interestingly, the remaining RPH1 putative paralogs (RPH1L2-5) serve as effectors that counteract RPH1-induced PTI, likely by disrupting the extracellular perception of RPH1. This research uncovers a HAMP whose homologs are conserved in both chewing and piercing-sucking insects. Moreover, it unveils an extracellular evasion mechanism utilized by herbivores to circumvent plant immunity using functionally differentiated paralogs., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Chloroplast Unusual Positioning 1 (CHUP1) plays an important role in the chloroplast avoidance and accumulation responses in mesophyll cells. In epidermal cells, prior research showed silencing CHUP1-induced chloroplast stromules and amplified effector-triggered immunity (ETI); however, the underlying mechanisms remain largely unknown. CHUP1 has a dual function in anchoring chloroplasts and recruiting chloroplast-associated actin (cp-actin) filaments for blue light-induced movement. To determine which function is critical for ETI, we developed an approach to quantify chloroplast anchoring and movement in epidermal cells. Our data show that silencing NbCHUP1 in Nicotiana benthamiana plants increased epidermal chloroplast de-anchoring and basal movement but did not fully disrupt blue light-induced chloroplast movement. Silencing NbCHUP1 auto-activated epidermal chloroplast defense (ECD) responses including stromule formation, perinuclear chloroplast clustering, the epidermal chloroplast response (ECR), and the chloroplast reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ). These findings show chloroplast anchoring restricts a multifaceted ECD response. Our results also show that the accumulated chloroplastic H 2 O 2 in NbCHUP1-silenced plants was not required for the increased basal epidermal chloroplast movement but was essential for increased stromules and enhanced ETI. This finding indicates that chloroplast de-anchoring and H 2 O 2 play separate but essential roles during ETI., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Phytophthora species, an oomycete plant pathogen, secrete effectors into plant cells throughout their life cycle for manipulating host immunity to achieve successful colonization. However, the molecular mechanisms underlying effector-triggered necrotic cell death remain elusive. In this study, we identified an RXLR (amino acid residue; Arginine-Any amino acid-Leucine-Arginine motif) effector (Pc12) from Phytophthora capsici, which contributes to virulence and induces necrosis by triggering a distinct endoplasmic reticulum (ER) stress response through its interaction with Rab13-2. The necrotic cell death induced by Pc12 did not exhibit conventional effector-triggered immunity-mediated hypersensitive cell death, including the involvement of nucleotide-binding site leucine-rich repeat downstream signaling components and transcriptional reprogramming of defense-related genes. Instead, it alters the localization of ER-resident proteins and confines secretory proteins within the ER. Pc12 directly interacts with Rab13-2, which is primarily localized to the ER and Golgi apparatus, resulting in a diminished Rab13-2 signal on the Golgi apparatus. Furthermore, Rab13-2 exhibits increased affinity for its interactor, Rab escort protein 1, in the presence of Pc12. Structural predictions revealed that a specific residue of Rab13-2 is crucial for binding to the C-terminus of Pc12. Substitution of this residue reduced its interaction with Pc12 and impaired P. capsici infection while maintaining its interaction with Rab escort protein 1 and prenylated Rab acceptor 1. These findings provide insight into how a pathogen effector induces a distinct form of necrotic cell death to facilitate colonization of the host plant by disrupting the recycling of Rab13-2, a protein involved in vesicle trafficking at the ER-Golgi interface., Competing Interests: DECLARATION OF COMPETING INTERESTS The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many gram-negative pathogenic bacteria use type III effector proteins (T3Es) as essential virulence factors to suppress host immunity and to cause disease. However, in many cases the molecular function of T3Es remains unknown. The plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on tomato and pepper plants and is known to translocate around 36 T3Es into its host cell, which collectively suppress plant defence and promote infection. XopM is an Xcv core T3E with unknown function that has no similarity to any other known protein. We found that XopM interacts with vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) in an isoform-specific manner. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of membrane contact sites involved in both tethering and lipid transfer by binding directly to proteins containing an FFAT (two phenylalanines [FF] in an acidic tract [AT]) motif. Sequence analyses revealed that XopM displays two FFAT motifs that cooperatively mediated the interaction of XopM with VAP. When expressed in plants, XopM supported growth of a nonpathogenic bacterial strain and dampened the production of reactive oxygen species, indicating its ability to suppress plant immunity. Further analyses revealed that the interaction with VAP and the ability to suppress microbe-associated molecular pattern-triggered immunity (MTI) are structurally and functionally separable, although XopM requires localisation to the host membrane system for full MTI suppression activity. We discuss a working model in which XopM uses FFAT motifs to target the membrane to interfere with early MTI responses., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

RNA silencing plays a crucial role in defending against viral infections in diverse eukaryotic hosts. Despite extensive studies on core components of the antiviral RNAi pathway such as DCLs, AGOs and RDRs proteins, host factors involved in antiviral RNAi remain incompletely understood. In this study, we employed the proximity labelling approach to identify the host factors required for antiviral RNAi in Nicotiana benthamiana. Using the barley stripe mosaic virus (BSMV)-encoded γb, a viral suppressor of RNA silencing (VSR), as the bait protein, we identified the DEAD-box RNA helicase RH20, a broadly conserved protein in plants and animals with a homologous human protein known as DDX5. We demonstrated the interaction between RH20 and BSMV γb. Knockdown or knockout of RH20 attenuates the accumulation of viral small interfering RNAs, leading to increased susceptibility to BSMV, while overexpression of RH20 enhances resistance to BSMV, a process requiring the cytoplasmic localization and RNA-binding activity of RH20. In addition to BSMV, RH20 also negatively regulates the infection of several other positive-sense RNA viruses, suggesting the broad-spectrum antiviral activity of RH20. Mechanistic analysis revealed the colocalization and interaction of RH20 with SGS3/RDR6, and disruption of either SGS3 or RDR6 undermines the antiviral function of RH20, suggesting RH20 as a new component of the SGS3/RDR6 bodies. As a counter-defence, BSMV γb VSR subverts the RH20-mediated antiviral defence by interfering with the RH20-SGS3 interaction. Our results uncover RH20 as a new positive regulator of antiviral RNAi and provide new potential targets for controlling plant viral diseases., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Key Message: Glycosylphosphatidylinositol-anchored protein (GPI-AP) Aa049 works as a key pathogenic factor to assist A. alternata in infecting plants, which is associated with the reactive oxygen species (ROS) pathway. Chrysanthemum black spot disease is a common fungal disease caused by A. alternata, which has severely hindered the development of the chrysanthemum industry. However, there are few reports on pathogenic factors in A. alternata, especially regarding GPI-APs. In this study, we identified a GPI-AP, Aa049, from A. alternata. Bioinformatics predictions suggest the presence of GPI-anchored modification sites at the C-terminus of its amino acid sequence, which is relatively conserved among different Alternaria Nees. Transient overexpression of Aa049 in Nicotiana benthamiana can induce programmed cell death (PCD), and the appearance of necrosis depends on its native signal peptide and GPI-anchored sites. Compared with the wild-type strain, the morphology and growth rate of the colony and mycelia of the ΔAa049-deletion mutants do not change. Still the integrity of the cell wall is damaged, and the virulence of the strain is significantly reduced, indicating that Aa049 plays an essential role as a pathogenic factor in the infection process of A. alternata. Furthermore, the results of quantitative real-time PCR (qRT-PCR) and physiological indicators suggested that the virulence of Aa049 may be exerted through the synthesis and clearance pathways of ROS. This study reveals that GPI-APs in A. alternata can act as virulence factors to aid pathogen invasion, providing a potential target for the development of future biopesticides., Competing Interests: Declarations Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Type 2C protein phosphatases (PP2Cs) constitute a large family in most plant species, but relatively few of them have been implicated in immunity. To identify and characterize PP2C phosphatases that affect tomato (Solanum lycopersicum) immunity, we generated loss-of-function mutations in 11 PP2C-encoding genes whose expression is altered in response to immune elicitors or pathogens. We report that 2 closely related PP2C phosphatases, PP2C immunity-associated candidate 3 (Pic3) and Pic12, are involved in regulating resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Loss-of-function mutations in Pic3 led to enhanced resistance to Pst in older but not younger leaves, whereas such mutations in Pic12 resulted in enhanced resistance in both older and younger leaves. Overexpression of Pic3 and Pic12 proteins in leaves of Nicotiana benthamiana inhibited resistance to Pst, and this effect was dependent on Pic3/12 phosphatase activity and an N-terminal palmitoylation motif associated with localization to the cell periphery. Pic3, but not Pic12, had a slight negative effect on flagellin-associated reactive oxygen species generation, although their involvement in the response to Pst appeared independent of flagellin. RNA-sequencing analysis of Rio Grande (RG)-PtoR wild-type plants and 2 independent RG-pic3 mutants revealed that the enhanced disease resistance in RG-pic3 older leaves is associated with increased transcript abundance of multiple defense-related genes. RG-pic3/RG-pic12 double-mutant plants exhibited stronger disease resistance than RG-pic3 or RG-pic12 single mutants. Together, our results reveal that Pic3 and Pic12 negatively regulate tomato immunity in an additive manner through flagellin-independent pathways., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Although microbial pathogens utilize various strategies to evade plant immunity, host plants have evolved powerful defense mechanisms that can be activated in preparation for threat by infective organisms. Here, we identified one 24 kDa alkyl hydroperoxide reductase C (AhpC) from the culture supernatant of Ralstonia solanacearum strain FQY-4 (denoted RsAhpC) in the presence of host roots. RsAhpC contributes to H 2 O 2 detoxification and the pathogenicity of R. solanacearum. However, the introduction of RsAhpC into the apoplast could activate immune defense, leading to suppression of pathogen colonization in both Nicotiana benthamiana and the Honghua Dajinyuan (HD) cultivar of N. tabacum. Consequently, overexpression of RsAhpC in the HD cultivar enhanced the resistance of tobacco to bacterial wilt disease caused by FQY-4. Overall, this study provides insight into the arms race between pathogens and their plant hosts. Specifically, it is firstly reported that plants can sense pathogen-derived AhpC to activate defenses, in addition to the role of AhpC in pathogen ROS detoxification. Therefore, the macromolecule AhpC produced by Ralstonia solanacearum has the ability to enhance plant defense as an elicitor, which provides a practical strategy for disease resistance breeding., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest or personal relationships that could have appeared to influence the work reported in the paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ciboria shiraiana is a necrotrophic fungus that causes mulberry sclerotinia disease resulting in huge economic losses in agriculture. During infection, the fungus uses immunity elicitors to induce plant tissue necrosis that could facilitate its colonization on plants. However, the key elicitors and immune mechanisms remain unclear in C. shiraiana. Herein, a novel elicitor Cs08297 secreted by C. shiraiana was identified, and it was found to target the apoplast in plants to induce cell death. Cs08297 is a cysteine-rich protein unique to C. shiraiana, and cysteine residues in Cs08297 were crucial for its ability to induce cell death. Cs08297 induced a series of defence responses in Nicotiana benthamiana, including the burst of reactive oxygen species (ROS), callose deposition, and activation of defence-related genes. Cs08297 induced-cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1. Purified His-tagged Cs08297-thioredoxin fusion protein triggered cell death in different plants and enhanced plant resistance to diseases. Cs08297 was necessary for sclerotial development, oxidative-stress adaptation, and cell wall integrity but negatively regulated virulence of C. shiraiana. In conclusion, our results revealed that Cs08297 is a novel fungal elicitor in fungi inducing plant immunity. Furthermore, its potential to enhance plant resistance provides a new target to control agricultural diseases biologically., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Blue light enhances the susceptibility of Nicotiana benthamiana to Phytophthora infestans, a causative agent of late blight disease. Investigating how blue light affects potato late blight resistance is an interesting aspect of exploring new ways to control late blight disease. Blue light photoreceptor phototropins (phot1, phot2) and their downstream interact protein StNRL1 have been shown to negatively regulate late blight resistance. In order to investigate whether other potato NPH3/RPT2-Like (NRL) family members are involved in regulating late blight resistance, this study focused on the potato NRL proteins containing RxSxS motif at the C-terminus. Another potato NRL protein StNRL-9, containing RxSxS motifs, was found to negatively regulate P. infestans resistance in potato and N. benthamiana. Overexpression of StNRL-9 in potato and N. benthamiana suppresses the accumulation of reactive oxygen species (ROS) and expression of the PTI marker genes NbWRKY7 and NbWRKY8. Similar to StNRL1, StNRL-9 interacts with the blue light receptors Stphot1 and Stphot2 on the cell membrane and could promote the degradation of a positive immune regulator StSWAP70. However StNRL-9 does not inhibit INF1-mediated cell death (ICD), which is different from the StNRL1 that inhibits ICD, indicating that both StNRL1 and StNRL-9 inhibit plant immunity in diverse ways. This study provides valuable information for further exploration of how plant phototropins and NRL family proteins regulate plant immunity., (© 2024 Scandinavian Plant Physiology Society.)

Puccinia striiformis f. sp. tritici (Pst) has a wide range and serious damage, which severely threatens global wheat production. In this study, we focused on an effector protein Pst3180.3, which was induced to be highly expressed during the Pst infection stage. The N-terminal 19 amino acid of Pst3180.3 was verified to function as a signal peptide and transferred to cytoplasm and nucleus of wheat following Pst infection. Transient overexpression of Pst3180.3 in Nicotiana benthamiana inhibited programmed cell death triggered via BAX. The instantaneous silencing of Pst3180.3 by BSMV- HIGS significantly reduced the number of uredinia and increased accumulation of reactive oxygen species. Those results indicated that Pst3180.3 is an important pathogenic factor of Pst. Interaction of Pst3180.3 with a transcription factor TaMYB4L in host was confirmed through yeast two-hybrid, luciferase complementation, and co-immunoprecipitation. Virus-induced gene silencing of TaMYB4L weakened the resistance to Pst, indicated that TaMYB4L may be involved in the positive regulation of plant immunity. Dual-luciferase assays revealed that Pst3180.3 inhibited the transcriptional activity of TaMYB4L. Meanwhile, molecular docking analysis identified the key residue sites for the interaction and binding between Pst3180.3 and MYB4L. Those results demonstrated that Pst3180.3 binds to TaMYB4L and interacts to inhibit wheat resistance to Pst infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) causes a devastating canker disease in yellow-fleshed kiwifruit (Actinidia chinensis). The effector HopZ5, which is present in all isolates of Psa3 causing global outbreaks of pandemic kiwifruit canker disease, triggers immunity in Nicotiana benthamiana and is not recognised in susceptible A. chinensis cultivars. In a search for N. benthamiana nonhost resistance genes against HopZ5, we found that the nucleotide-binding leucine-rich repeat receptor NbPTR1 recognised HopZ5. RPM1-interacting protein 4 orthologues from N. benthamiana and A. chinensis formed a complex with NbPTR1 and HopZ5 activity was able to disrupt this interaction. No functional orthologues of NbPTR1 were found in A. chinensis. NbPTR1 transformed into Psa3-susceptible A. chinensis var. chinensis 'Hort16A' plants introduced HopZ5-specific resistance against Psa3. Altogether, this study suggested that expressing NbPTR1 in Psa3-susceptible kiwifruit is a viable approach to acquiring resistance to Psa3 and it provides valuable information for engineering resistance in otherwise susceptible kiwifruit genotypes., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Non-host resistance (NHR) governs defense responses against a broad range of potential pathogen species in contrast with host resistance. To identify specific genes involved in disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana and identified glycosyltransferase (NbGT) as an essential component of NHR. NbGT silencing enhanced the hypersensitivity response, reactive oxygen species response, and callose deposition in N. benthamiana, improving its NHR to Pseudomonas syringae pv. tomato (Pst) DC3000. NbGT participated in reactive oxygen species accumulation caused by flg22 rather than coronatine and HrcC of Pst DC3000. Analyses of gene expression and enzyme activity demonstrated that NbGT-silenced plants exhibited enhanced expression and elevated levels of superoxide dismutase, resulting in heightened accumulation of H 2 O 2 . In conclusion, NbGT-silencing increases H 2 O 2 accumulation by regulating superoxide dismutase activity during the immune response to flg22, enhancing resistance to Pst DC3000 in N. benthamiana. This research provides novel insights into the role of glycosyltransferases in NHR., (© 2024 Scandinavian Plant Physiology Society.)

Obligate biotrophic powdery mildew fungi infect a wide range of economically important plants. These fungi often deliver effector proteins into the host tissues to suppress plant immunity and sustain infection. The phytohormone salicylic acid (SA) is one of the most important signals that activate plant immunity against pathogens. However, how powdery mildew effectors interact with host SA signalling is poorly understood. Isochorismatase (ISC) effectors from two other filamentous pathogens have been found to inhibit host SA biosynthesis by hydrolysing isochorismate, the main SA precursor in the plant cytosol. Here, we identified an ISC effector, named EqIsc1, from the rubber tree powdery mildew fungus Erysiphe quercicola. In ISC enzyme assays, EqIsc1 displayed ISC activity by transferring isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate in vitro and in transgenic Nicotiana benthamiana plants. In EqIsc1-expressing transgenic Arabidopsis thaliana, SA biosynthesis and SA-mediated immune response were significantly inhibited. In addition, we developed an electroporation-mediated transformation method for the genetic manipulation of E. quercicola. Inoculation of rubber tree leaves with EqIsc1-silenced E. quercicola strain induced SA-mediated immunity. We also detected the translocation of EqIsc1 into the plant cytosol during the interaction between E. quercicola and its host. Taken together, our results suggest that a powdery mildew effector functions as an ISC enzyme to hydrolyse isochorismate in the host cytosol, altering the SA biosynthesis and immune response., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 from Nicotiana benthamiana accumulates in vivo as a homodimer that converts into a higher-order oligomer upon activation by its upstream virus disease resistance protein Rx. The cryo-EM structure of NbNRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation and contribute to immune receptor autoinhibition. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways, possibly to minimise undesired cross-activation and evade pathogen suppression of immunity. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes., Competing Interests: TOB and SK receive funding from industry on NLR biology and cofounded a start-up company (Resurrect Bio Ltd.) on resurrecting disease resistance. JK, LD, SK and MPC have filed patents on NLR biology. LD and MPC have received fees from Resurrect Bio Ltd., (Copyright: © 2024 Selvaraj et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Producing recombinant proteins in plants has become a valuable alternative to traditional microbial or mammalian systems due to its cost-effectiveness, scalability, and ability to perform post-translational modifications. This study investigates the use of the Tobacco Mosaic Virus (TMV)-based vector system for producing the Dengue virus serotype 3 (DENV-3) envelope domain III (EDIII) protein in plants.. A fragment of the gene that encodes domain III of the dengue 3 envelope protein (D3EIII, comprising 300-420 amino acids), was effectively expressed within Nicotiana tabacum plants utilizing a transient expression system based on tobacco mosaic virus (TMV). The N-terminal 5' UTR region upstream of D3EIII notably enhanced protein yield in infected tissues. The produced recombinant protein exhibited reactivity with both (anti) D3EIII polyclonal antibodies and antibodies of anti-His tag. Upon injection of EDIII in mice, it stimulated the generation of antibodies against the dengue-specific virus. The induced antibodies demonstrated neutralizing activity against dengue virus type 3. These findings indicate that the TMV expression system is effective for producing dengue virus antigens in plants, resulting in antigens with appropriate properties and strong immunogenic potential.

Persistent plant viruses are widespread in natural ecosystems. However, little is known about why persistent infection with these viruses may cause little or no harm to their host. Here, we discovered a new polerovirus that persistently infected wild rice plants by deep sequencing and assembly of virus-derived small-interfering RNAs (siRNAs). The new virus was named Rice tiller inhibition virus 2 (RTIV2) based on the symptoms developed in cultivated rice varieties following Agrobacterium-mediated inoculation with an infectious RTIV2 clone. We showed that RTIV2 infection induced antiviral RNA interference (RNAi) in both the wild and cultivated rice plants as well as Nicotiana benthamiana. It is known that virulent virus infection in plants depends on effective suppression of antiviral RNAi by viral suppressors of RNAi (VSRs). Notably, the P0 protein of RTIV2 exhibited weak VSR activity and carries alanine substitutions of two amino acids broadly conserved among diverse poleroviruses. Mixed infection with umbraviruses enhanced RTIV2 accumulation and/or enabled its mechanical transmission in N. benthamiana. Moreover, replacing the alanine at either one or both positions of RTIV2 P0 enhanced the VSR activity in a co-infiltration assay, and RTIV2 mutants carrying the corresponding substitutions replicated to significantly higher levels in both rice and N. benthamiana plants. Together, our findings show that as a persistent plant virus, RTIV2 carries specific mutations in its VSR gene to weaken viral suppression of antiviral RNAi. Our work reveals a new strategy for persistent viruses to maintain long-term infection by weak suppression of the host defence response., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Autophagy, an intracellular degradation process, has emerged as a crucial innate immune response against various plant pathogens, including viruses. Tomato spotted wilt orthotospovirus (TSWV) is a highly destructive plant pathogen that infects over 1000 plant species and poses a significant threat to global food security. However, the role of autophagy in defence against the TSWV pathogen, and whether the virus counteracts this defence, remains unknown. In this study, we report that autophagy plays an important role in antiviral defence against TSWV infection; however, this autophagy-mediated defence is counteracted by the viral effector NSs. Transcriptome profiling revealed the up-regulation of autophagy-related genes (ATGs) upon TSWV infection. Blocking autophagy induction by chemical treatment or knockout/down of ATG5/ATG7 significantly enhanced TSWV accumulation. Notably, the TSWV nucleocapsid (N) protein, a major component of the viral replication unit, strongly induced autophagy. However, the TSWV nonstructural protein NSs was able to effectively suppress N-induced autophagy in a dose-dependent manner. Further investigation revealed that NSs inhibited ATG6-mediated autophagy induction. These findings provide new insights into the defence role of autophagy against TSWV, a representative segmented negative-strand RNA virus, as well as the tospoviral pathogen counterdefence mechanism., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

MicroRNA-like RNAs (milRNAs) play a significant role in the infection process by plant-pathogenic fungi. However, the specific functions and regulatory mechanisms of fungal milRNAs remain insufficiently elucidated. This study investigated the function of Foc-milR138, an infection-induced milRNA secreted by Fusarium oxysporum f. sp. cubense (Foc), which is the causal agent of Fusarium wilt of banana. Initially, through precursor gene knockout and phenotypic assessments, we confirmed that Foc-milR138 acts as a virulent milRNA prominently upregulated during the early stages of Foc infection. Subsequent bioinformatic analyses and transient expression assays in Nicotiana benthamiana leaves identified a host receptor-like kinase gene, MaLYK3, as the direct target of Foc-milR138. Functional investigations of MaLYK3 revealed its pivotal role in triggering immune responses of N. benthamiana by upregulating a suite of resistance genes, bolstering reactive oxygen species (ROS) accumulation and callose deposition, thereby fortifying disease resistance. This response was markedly subdued upon co-expression with Foc-milR138. Expression pattern analysis further verified the specific suppression of MaLYK3 by Foc-milR138 during the early root infection by Foc. In conclusion, Foc secretes a virulent milRNA (Foc-milR138) to enter the host banana cells and inhibit the expression of the plant surface receptor-like kinase MaLYK3, subverting the disease resistance activated by MaLYK3, and ultimately facilitating pathogen invasion. These findings shed light on the roles of fungal milRNAs and their targets in resistance and pathogenicity, offering promising avenues for the development of disease-resistant banana cultivars., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Competing Interests: Conflict of interest statement. None declared.

Hypersensitive response-programmed cell death (HR-PCD) regulated by Ca 2+ signal is considered the major regulator of resistance against Puccinia triticina (Pt.) infection in wheat. In this study, the bread wheat variety Thatcher and its near-isogenic line with the leaf rust resistance locus Lr26 were infected with the Pt. race 260 to obtain the compatible and incompatible combinations, respectively. The expression of translationally controlled tumor protein (TaTCTP) was upregulated upon infection with Pt., through a Ca 2+ -dependent mechanism in the incompatible combination. The knockdown of TaTCTP markedly increased the area of dying cell and the number of Pt. haustorial mother cells (HMCs) at the infection sites, whereas plants overexpressing the gene exhibited enhanced resistance. The interaction between TaTCTP and calcineurin B-like protein-interacting protein kinase 23 (TaCIPK23) was also investigated, and the interaction was found occurred in the endoplasmic reticulum. TaCIPK23 phosphorylated TaTCTP in vitro. The expression of a phospho-mimic TaTCTP mutant in Nicotiana benthamiana promoted HR-like cell death. Silencing TaCIPK23 or TaCIPK23/TaTCTP co-silencing resulted in the same results as silencing TaTCTP. This suggested that TaTCTP is a novel phosphorylation target of TaCIPK23, and both participate in the resistance of wheat to Pt. in the same pathway., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Bursaphelenchus xylophilus (pine wood nematode, PWN), a migratory plant-parasitic nematode, acts as an etiological agent, inflicting considerable damage to pine forests worldwide. Plant immunity constitutes a crucial factor in resisting various pathogenic invasions. The primary defensive responses of host pines against PWN infection encompass terpene accumulation, defense response-related gene expression, and programmed cell death. Venom allergen-like proteins (VAPs), as potential effectors, are instrumental in facilitating the successful colonization of PWNs. In this study, we investigated the inhibition of B. xylophilus VAP ( BxVAP1 ) expression by RNA interference in vitro. Following BxVAP1 silencing, the reproduction rate and migration rate of the PWN population in Pinus massoniana decreased, the expression of the α-pinene synthase gene was induced, other terpene synthase and pathogenesis-related genes were inhibited and delayed, the peak times and levels of terpene-related substances were changed, and the degree of cavitation in P. massoniana was diminished. Transient expression of BxVAP1 in Nicotiana benthamiana revealed that BxVAP1 was expressed in both the cell membrane and nucleus, inducing programmed cell death and the expression of pathogen-associated molecular pattern-triggered immunity marker genes ( NbAcre31 and NbPTI5 ). This study is the first to demonstrate that silencing the BxVAP1 gene affects host defense responses, including terpenoid metabolism in P. massoniana , and that BxVAP1 can be recognized by N. benthamiana as an effector to trigger its innate immunity, expanding our understanding of the parasitic mechanism of B. xylophilus ., Competing Interests: The author(s) declare no conflict of interest.

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors' ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection and suggests that, similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonization., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)