Peroxisomes are ubiquitous organelles that play vital roles in various physiological and biochemical processes, including fatty acid β-oxidation and reactive oxygen species (ROS) metabolism. These organelles have been implicated in the pathogenicity of many plant fungal pathogens. In this study, CaPex8, a homolog of Saccharomyces cerevisiae Pex8, was identified and characterized in Colletotrichum aenigma. CaPEX8 was found to localize to peroxisomes, and its deletion impaired the mutant’s ability to utilize fatty acids as a carbon source. Using a green fluorescent protein (GFP) fused to the peroxisomal targeting signal PTS1, the import of peroxisomal matrix proteins was shown to be defective in ΔCapex8 mutants. Additionally, the mutants exhibited elevated conidiation, increased sensitivity to osmotic stress and oxidative stress, and impaired cell wall integrity. Peroxisome biogenesis was also disrupted in the absence of CaPEX8. Taken together, these results demonstrate that CaPex8 is essential for maintaining peroxisomal structure and function, and it significantly influences fungal growth, development, and pathogenicity in C. aenigma.

Satellite RNAs (satRNAs) are parasitic subviral agents whose biological activities are mediated largely by specific sequence determinants and structured RNA elements. Establishing biologically supported global RNA structures is therefore essential for understanding how RNA architecture underlies satRNA functions. Cucumber mosaic virus (CMV) satRNA is one of the best-characterized models for investigating satRNA structure-function relationships; however, a biologically supported global RNA architecture of CMV satRNA has yet to be established. Here, we applied AlphaFold3 modeling to predict the global structure of CMV satRNA T1 (sat-T1). Initial full-length structure modeling generated multiple long-distance interactions that lacked biological support. We therefore used fragment-based modeling, combined with chemical probing, evolutionary covariation, and compensatory mutagenesis, to derive a biologically supported global secondary structure. To determine whether structurally distant regions could interact in the context of the full-length RNA, we engineered a structure-guided T1-ZD mutant that preserved the supported secondary structure while reducing alternative base-pairing possibilities. Full-length AlphaFold3 modeling of T1-ZD largely recapitulated the proposed architecture, while one predicted model revealed a long-distance interaction that was subsequently supported by compensatory mutagenesis analysis. These findings suggest that the 3′ terminus of sat-T1 may undergo conformational switching between alternative structural states. Together, our work establishes a biologically supported global RNA architecture for CMV sat-T1 and provides a structural framework for investigating the molecular basis of satRNA function.

Nonsense-mediated mRNA decay (NMD) is a critical RNA quality control system in eukaryotes, also playing a role in defending against viral infections. However, research has primarily focused on nonsegmented viruses. To investigate the interaction between NMD and segmented RNA viruses, we used cucumber mosaic virus (CMV), which possesses a tripartite, single-stranded, positive-sense RNA genome. Agroinfiltration assays were performed to assess how CMV RNA segments, or their variants, respond to NMD. We found that CMV genomic segments (RNAs 1-3) exhibit distinct responses to NMD. Specifically, RNA3, which serves as the translation template of the movement protein (MP), is selectively degraded by NMD, unlike RNA1 and RNA2, which encode viral replicase components. This degradation is triggered by the coat protein (CP) sequence and can be mitigated by the trans-expression of the 1a replicase or CP. The 1a protein requires its specific interaction with the Box-B motif of RNA3 to avoid NMD. Importantly, compromising NMD reduces CMV infection during the early stages, suggesting that NMD-mediated RNA3 degradation facilitates initial viral replication. This is supported by observations that MP expression in trans negatively regulates viral RNA replication. We propose a model to illustrate the molecular interplay between NMD and CMV, emphasising the implications of genomic segmentation in NMD-virus interactions.

In plants, small peptides are important players in the plant stress response, yet their function in plant antiviral responses remains poorly understood. Here, we identify that the plant small peptide, CLAVATA3/ESR-RELATED 7 (CLE7), enhances plant resistance to Chinese wheat mosaic virus infection in Nicotiana (N.) benthamiana. Subsequent investigations demonstrate that CLE7 recognizes receptor kinase NbPXC3 to control the plant antiviral response. Moreover, CLE7-NbPXC3 signaling induces NbMKK2-controlled NbMPK4 phosphorylation, resulting in phosphorylation of the transcription factor NbEDT1. NbEDT1 phosphorylation is involved in the transcriptional activity of NbNCED3, which is a rate-limiting enzyme in abscisic acid (ABA) biosynthesis. Moreover, CLE7 activates broad-spectrum disease resistance to multiple RNA viral infections. Our study indicates that CLE7 induces a plant antiviral response through a series of immune signal transductions in N. benthamiana and provides a foundation for the exploration of efficient viral disease management methods based on plant small peptides.

In this study, the insert length, location within the coat protein-encoding gene, and sequence orientation of the target fragment were optimized to construct an efficient virus-induced gene silencing (VIGS) system in melon using a Begomovirus solanumdelhiense vector. Existing systems are mostly RNA viruses, requiring in vitro synthesis of viral strands that are prone to degradation, although they exhibit high infectivity and stability in cucurbit hosts and ease of manipulation. This vector was selected for its more stable genome structure and these advantages. The melon phytoene desaturase (CmPDS), a key gene of carotenoid biosynthesis, was selected as a reporter gene to evaluate the effects of the VIGS system. Our results revealed that the melon leaves in all the VIGS treatments exhibited a typical photobleaching phenotype at 21 days post-inoculation. Moreover, reverse transcription quantitative real-time PCR revealed a significant reduction in the mRNA levels of PDS in melon. The highest silencing efficiency (lowest PDS mRNA levels) was achieved by the VIGS vector harboring a 165 bp CmPDS fragment at the 3′ end of the AV1. These findings not only establish a more efficient VIGS protocol for melon but also provide a foundation for developing novel virus-based silencing tools applicable to functional genomics and cucurbit crop improvement, particularly for traits requiring precise gene expression modulation such as disease resistance and fruit quality.

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As a type of parasitic agent, satellite RNAs (satRNAs) rely on cognate helper viruses to achieve their replication and transmission. During the infection of satRNAs, helper virus RNAs serve as templates for synthesizing viral proteins, including the replication proteins essential for satRNA replication. However, the role of non-template functions of helper virus RNAs in satRNA replication remains unexploited. Here we employed the well-studied model that is composed of cucumber mosaic virus (CMV) and its associated satRNA. In the experiments employing the CMV trans-replication system, we observed an unexpected phenomenon the replication proteins of the mild strain LS-CMV exhibited defective in supporting satRNA replication, unlike those of the severe strain Fny-CMV. Independent of translation products, all CMV genomic RNAs could enhance satRNA replication, when combined with the replication proteins of CMV. This enhancement is contingent upon the recruitment and complete replication of helper virus RNAs. Using the method developed for analyzing the satRNA recruitment, we observed a markedly distinct ability of the replication proteins from both CMV strains to recruit the positive-sense satRNA-harboring RNA3 mutant for replication. This is in agreement with the differential ability of both 1a proteins in binding satRNAs in plants. The discrepancies provide a convincing explanation for the variation of the replication proteins of both CMV strains in replicating satRNAs. Taken together, our work provides compelling evidence that the non-template functions of helper virus RNAs create an optimal replication environment to enhance satRNA proliferation.

Nucleotide-binding leucine-rich repeat (NLR) proteins contribute widely to plant immunity by regulating defense mechanisms through the elicitation of a hypersensitive response (HR). Here, we find that TaRACK1B (the receptor for activated C-kinase 1B) regulates wheat immune response against Chinese wheat mosaic virus (CWMV) infection. TaRACK1B recruits TaSGT1 and TaHSP90 to form the TaRACK1B-TaSGT1-TaHSP90 complex. This complex is essential for maintaining NLR proteins’ stability (TaRGA5-like and TaRGH1A-like) in order to control HR activation and inhibit viral infection. However, the cysteine-rich protein encoded by CWMV can disrupt TaRACK1B-TaSGT1-TaHSP90 complex formation, leading to the reduction of NLR-protein stability and suppression of HR activation, thus promoting CWMV infection. Interestingly, the 7K protein of wheat yellow mosaic virus also interferes with this antiviral immunity. Our findings show a shared viral counter-defense strategy whereby two soil-borne viruses may disrupt the TaRACK1B-TaSGT1-TaHSP90 complex, suppressing NLR-protein-mediated broad-spectrum antiviral immunity and promoting viral infection in wheat.

Tomato spotted wilt virus-mediated delivery of CRISPR/Cas9 bypasses the need for stable transformation and permits efficient, DNA-free genome editing in pepper. Remarkably, up to 77.9% of regenerated pepper plants contained heritable edits. This method has been validated with two pepper varieties and is compatible with existing tissue culture protocols.

The necrogenic strain N5 of tomato mosaic virus (ToMV-N5) causes systemic necrosis in tomato cultivar Hezuo903. In this work, we mapped the viral determinant responsible for the induction of systemic necrosis. By exchanging viral genes between N5 and a non-necrogenic strain S1, we found that movement protein (MP) was the determinant for the differential symptoms caused by both strains. Compared with S1 MP, N5 MP had an additional ability to increase virus accumulation, which was not due to its functions in viral cell-to-cell movement. Actually, N5 MP, but not S1 MP, was a weak RNA silencing suppressor, which assisted viral accumulation. Sequence alignment showed that both MPs differed by only three amino acid residues. Experiments with viruses having mutated MPs indicated that the residue isoleucine at position 170 in MP was the key site for MP to increase virus accumulation, but also was required for MP to induce systemic necrosis in virus-infected tomato plants. Collectively, the lethal necrosis caused by N5 is dependent on its MP protein that enhances virus accumulation via its RNA silencing suppressor activity, probably leading to systemic necrosis responses in tomato plants.

Southern rice black-streaked dwarf virus (SRBSDV; Fijivirus, Reoviridae) has become a threat to cereal production in East Asia in recent years. Our previous cytopathologic studies have suggested that SRBSDV induces a process resembling programmed cell death in infected tissues that results in distinctive growth abnormalities. The viral product responsible for the cell death, however, remains unknown. Here P9-2 protein, but not its RNA, was shown to induce cell death in Escherichia coli and plant cells when expressed either locally with a transient expression vector or systemically using a heterologous virus. Both computer prediction and fluorescent assays indicated that the viral nonstructural protein was targeted to the plasma membrane (PM) and further modification of its subcellular localization abolished its ability to induce cell death, indicating that its PM localization was required for the cell death induction. P9-2 was predicted to harbour two transmembrane helices within its central hydrophobic domain. A series of mutation assays further showed that its central transmembrane hydrophobic domain was crucial for cell death induction and that its conserved F90, Y101, and L103 amino acid residues could play synergistic roles in maintaining its ability to induce cell death. Its homologues in other fijiviruses also induced cell death in plant and bacterial cells, implying that the fijiviral nonstructural protein may trigger cell death by targeting conserved cellular factors or via a highly conserved mechanism.

A large number of viral delivery systems have been developed for characterizing functional genes and producing heterologous recombinant proteins in plants, and but most of them are unable to co-express two fusion-free foreign proteins in the whole plant for extended periods of time. In this study, we modified tobacco rattle virus (TRV) as a TRVe dual delivery vector, using the strategy of gene substitution. The reconstructed TRVe had the capability to simultaneously produce two fusion-free foreign proteins at the whole level of Nicotiana benthamiana, and maintained the genetic stability for the insert of double foreign genes. Moreover, TRVe allowed systemic expression of two foreign proteins with the total lengths up to ∼900 aa residues. In addition, Cas12a protein and crRNA were delivered by the TRVe expression system for site-directed editing of genomic DNA in N. benthamiana 16c line constitutively expressing green fluorescent protein (GFP). Taker together, the TRV-based delivery system will be a simple and powerful means to rapidly co-express two non-fused foreign proteins at the whole level and facilitate functional genomics studies in plants.

Nigrospora nonsegmented RNA virus 1 (NoNRV1) has been reported previously in the fungus Nigrospora oryzae, but its biological effects on its host are unknown. In this work, we isolated a strain 9-1 of N. oryzae from a chrysanthemum leaf and identified NoNRV1 infection in the isolated strain. The genome sequence of NoNRV1 identified here is highly homologous to that of the isolate HN-21 of NoNRV1 previously reported; thus, we tentatively designated the newly identified NoNRV1 as NoNRV1-ZJ. Drug treatment with Ribavirin successfully removed NoNRV1-ZJ from the strain 9-1, which provided us with an ideal control to determine the biological impacts of NoNRV1 infection on host fungi. By comparing the virus-carrying (9-1) and virus-cured (9-1C) strains, our results indicated that infection with NoNRV1 promoted the pigmentation of the host cells, while it had no discernable effects on host growth on potato dextrose agar plates when subjected to osmotic or oxidative stress. Interestingly, we observed inhibitory impacts of virus infection on the thermotolerance of N. oryzae and the pathogenicity of the host fungus in cotton leaves. Collectively, our work provides clear evidence of the biological relevance of NoNRV1 infection in N. oryzae, including pigmentation, hypovirulence, and thermotolerance.

In order to construct a viral vector that can simultaneously express two non-fusionproteins in the whole host plants.MethodsAgrobacteriuminfectious clone pYL156containing tobacco rattlevirus(TRV)genomic RNA2was used to construct dual expression vector pTRV2e2by deleting279bp of5' end of2bgene,changing initiation codon of2bgene ATG to AGG,and introducing the subgenomic promoter of peaearly-browning virus(PEBV)coat protein(cp)gene.Different exogenous genes were cloned into the downstreamof2band PEBVcpsubgenomic promoters to measure the ability of virus TRVe2to express two foreign proteins,assess the stability of reconstructed TRVe2and analyze the function of proteins in the seedlings ofNicotianabenthamiana.ResultsTRVe2could simultaneously and rapidly produce two non-fused target proteins andexpress at least a70ku foreign protein in the whole host plants;TRVe2harboring2.0kb exogenous gene couldstably exist inN.benthaminaplants and could be served as technical means for analyzing the biological functionof the proteins and the interaction between two proteins.ConclusionRecombinant virus TRVe2constructed inthis study provide a toolbox for fast and efficient production of double foreign proteins and for analysis of theinteraction between two proteins in the host plants

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

稻瘟病是一种由子囊真菌稻瘟病菌引起的危害粮食安全的世界性病害.多数效应蛋白对于稻瘟病菌的毒力起到重要作用,在与寄主植物的相互作用中至关重要,因此,筛选并鉴定稻瘟病菌效应蛋白,阐释其基因功能对于稻瘟病的防治具有重要意义.本研究根据已有的稻瘟病菌效应蛋白相关研究方法以及生物信息学软件筛选出了一个几丁质结合蛋白Mo-Chtbp1,进一步利用叶鞘细胞—效应子分泌荧光表达观察体系确定MoChtbp1是一个质外体效应子.利用同源重组的方法敲除MoChtBP1,发现MoChtBP1的缺失不会影响稻瘟病菌在生长、产孢以及附着胞形成等方面的能力,但会导致其致病能力明显减弱.本文结果将为新的抗病水稻选种育种提供实验材料及理论依据.

AbstractThe Chinese wheat mosaic virus (CWMV) genome consists of two positive‐strand RNAs that are required for CWMV replication and translation. The eukaryotic translation elongation factor (eEF1A) is crucial for the elongation of protein translation in eukaryotes. Here, we show that silencing eEF1A expression in Nicotiana benthamiana plants by performing virus‐induced gene silencing can greatly reduce the accumulation of CWMV genomic RNAs, whereas overexpression of eEF1A in plants increases the accumulation of CWMV genomic RNAs. In vivo and in vitro assays showed that eEF1A does not interact with CWMV RNA‐dependent RNA polymerase. Electrophoretic mobility shift assays revealed that eEF1A can specifically bind to the 3ʹ‐untranslated region (UTR) of CWMV genomic RNAs. By performing mutational analyses, we determined that the conserved region in the 3ʹ‐UTR of CWMV genomic RNAs is necessary for CWMV replication and translation, and that the sixth stem‐loop (SL‐6) in the 3ʹ‐UTR of CWMV genomic RNAs plays a key role in CWMV infection. We conclude that eEF1A is an essential host factor for CWMV infection. This finding should help us to develop new strategies for managing CWMV infections in host plants.

Pyricularia oryzae is the causal agent of blast disease on staple gramineous crops. Sulphur is an essential element for the biosynthesis of cysteine and methionine in fungi. Here, we targeted the P. oryzae PoMET3 encoding the enzyme ATP sulfurylase, and PoMET14 encoding the APS (adenosine-5′-phosphosulphate) kinase that are involved in sulfate assimilation and sulphur-containing amino acids biosynthesis. In P. oryzae, deletion of PoMET3 or PoMET14 separately results in defects of conidiophore formation, significant impairments in conidiation, methionine and cysteine auxotrophy, limited invasive hypha extension, and remarkably reduced virulence on rice and barley. Furthermore, the defects of the null mutants could be restored by supplementing with exogenous cysteine or methionine. Our study explored the biological functions of sulfur assimilation and sulphur-containing amino acids biosynthesis in P. oryzae.

As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.

半胱氨酸蛋白酶(cysteine proteinase,CysP)是一类重要的蛋白酶家族,广泛参与植物多种生理过程.为了分析植物CysP的特性,本实验首先通过RT-PCR技术从本氏烟中扩增获得了一个编码CysP的基因(Nb-CysP)序列并连接至pEASYTM-T5 Zero载体,测序验证后亚克隆至原核表达载体pGEX-6P1,命名为pGEX-NbCysP;将其导入大肠埃希菌BL21plysS中诱导表达;重组表达的NbCysP融合蛋白经过亲和层析纯化,免疫兔子制备多克隆抗体,Western印迹分析显示,该抗体能和重组NbCysP蛋白发生强烈的免疫学反应且条带单一,表明所获得的CysP抗体具有良好的特异性.上述结果为进一步鉴定植物CysP的功能特性奠定了基础.