ABSTRACT
Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.
Subject(s)
Carrier Proteins , Comovirus , Nicotiana , Plant Diseases , Plant Proteins , Vigna , Comovirus/metabolism , Comovirus/physiology , Comovirus/genetics , Vigna/virology , Vigna/metabolism , Nicotiana/virology , Nicotiana/metabolism , Nicotiana/genetics , Carrier Proteins/metabolism , Carrier Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Plant Diseases/virology , Cysteine Proteases/metabolism , Cysteine Proteases/genetics , Plants, Genetically Modified , Viral Proteins/metabolism , Viral Proteins/genetics , Capsid Proteins/metabolism , Capsid Proteins/genetics , Potyvirus/physiology , Potyvirus/metabolism , EndopeptidasesABSTRACT
The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection, environmental stress, and growth circumstances cause phosphorylation or dephosphorylation of plant initiation factors. Our findings indicate that casein kinase 2 can phosphorylate recombinant wheat eIFiso4E and eIFiso4G generated from E. coli in vitro. For wheat eIFiso4E, Ser-207 was found to be the in vitro phosphorylation site. eIFiso4E lacks an amino acid that can be phosphorylated at the position corresponding to Ser-209, the phosphorylation site in mammalian eIF4E, yet phosphorylation of eIFiso4E has effects on VPg binding affinity that are similar to those of phosphorylation of mammalian eIF4E. The addition of VPg and phosphorylated eIFiso4F to depleted wheat germ extract (WGE) leads to enhancement of translation of both uncapped and capped viral mRNA. The addition of PABP together with eIFiso4Fp and eIF4B to depleted WGE increases both uncapped and capped mRNA translation. However, it exhibits a translational advantage specifically for uncapped mRNA, implying that the phosphorylation of eIFiso4F hinders cap binding while promoting VPg binding, thereby facilitating uncapped translation. These findings indicate TEV virus mediates VPg-dependent translation by engaging a mechanism entailing phosphorylated eIFiso4Fp and PABP. To elucidate the molecular mechanisms underlying these observed effects, we studied the impact of PABP and/or eIF4B on the binding of VPg with eIFiso4Fp. The inclusion of PABP and eIF4B with eIFiso4Fp resulted in about 2-fold increase in affinity for VPg (Kd = 24 ± 1.7 nM), as compared to the affinity of eIFiso4Fp alone (Kd = 41.0 ± 3.1 nM). The interactions between VPg and eIFiso4Fp were determined to be both enthalpically and entropically favorable, with the enthalpic contribution accounting for 76-97% of the ΔG at 25°C, indicating a substantial role of hydrogen bonding in enhancing the stability of the complex. The binding of PABP to eIFiso4Fp·4B resulted in a conformational alteration, leading to a significant enhancement in the binding affinity to VPg. These observations suggest PABP enhances the affinity between eIFiso4Fp and VPg, leading to an overall conformational change that provides a stable platform for efficient viral translation.
Subject(s)
Eukaryotic Initiation Factors , Poly(A)-Binding Proteins , Potyvirus , Protein Binding , Protein Biosynthesis , Triticum , Phosphorylation , Potyvirus/metabolism , Potyvirus/genetics , Triticum/virology , Triticum/metabolism , Triticum/genetics , Eukaryotic Initiation Factors/metabolism , Eukaryotic Initiation Factors/genetics , Poly(A)-Binding Proteins/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/genetics , Casein Kinase II/metabolism , Casein Kinase II/geneticsABSTRACT
Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana-Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.
Subject(s)
Potyvirus , Viral Proteins , Viral Proteins/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , Potyvirus/metabolism , Plant DiseasesABSTRACT
Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.
Subject(s)
Amino Acids , Potyvirus , Amino Acid Sequence , Amino Acid Substitution/genetics , Amino Acids/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Plant Diseases/genetics , Potyvirus/genetics , Potyvirus/metabolism , Citrullus/virologyABSTRACT
Potato virus Y (PVY) is one of the most dangerous agricultural pathogens that causes substantial harm to vegetative propagated crops, such as potatoes (Solanum tuberosum L.). A necessary condition for PVY infection is an interaction between the plant cap-binding translation initiation factors eIF4E and a viral protein VPg, which mimics the cap-structure. In this study, we identified the point mutations in potato eIF4E1 and eIF4E2 that disrupt VPg binding while preserving the functional activity. For the structural interpretation of the obtained results, molecular models of all the studied forms of eIF4E1 and eIF4E2 were constructed and analyzed via molecular dynamics. The results of molecular dynamics simulations corresponds to the biochemical results and suggests that the ß1ß2 loop plays a key role in the stabilization of both eIF4E-cap and eIF4E-VPg complexes.
Subject(s)
Potyvirus , Solanum tuberosum , Solanum , Solanum/metabolism , Solanum tuberosum/genetics , Potyvirus/genetics , Potyvirus/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Plant Diseases/geneticsABSTRACT
Potyvirids, members of the family Potyviridae, produce the P3N-PIPO protein, which is crucial for the cell-to-cell transport of viral genomic RNAs. The production of P3N-PIPO requires an adenine (A) insertion caused by RNA polymerase slippage at a conserved GAAAAAA (GA6) sequence preceding the PIPO open reading frame. Presently, the slippage rate of RNA polymerase has been estimated in only a few potyvirids, ranging from 0.8 to 2.1%. In this study, we analyzed publicly available plant RNA-seq data and identified 19 genome contigs from 13 distinct potyvirids. We further investigated the RNA polymerase slippage rates at the GA6 motif. Our analysis revealed that the frequency of the A insertion variant ranges from 0.53 to 4.07% in 11 potyviruses (genus Potyvirus). For the two macluraviruses (genus Macluravirus), the frequency of the A insertion variant was found to be 0.72% and 10.96% respectively. Notably, the estimated RNA polymerase slippage rates for 12 out of the 13 investigated potyvirids were reported for the first time in this study. Our findings underscore the value of plant RNA-seq data for quantitative analysis of potyvirid genome variants, specifically at the GA6 slippage site, and contribute to a more comprehensive understanding of the RNA polymerase slippage phenomenon in potyvirids.
Subject(s)
Potyvirus , Viral Proteins , Viral Proteins/genetics , Viral Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Potyvirus/genetics , Potyvirus/metabolism , Genome, Viral , Open Reading Frames , RNA, Plant , RNA, Viral/genetics , Plant Diseases , PhylogenyABSTRACT
The viability of viral-derived nanoparticles (virions and VLPs) aimed to nanobiotechnological functionalizations of the coat protein (CP) of turnip mosaic virus has been studied by means of advanced computational methodologies that include molecular dynamics. The study has allowed to model the structure of the complete CP and its functionalization with three different peptides and obtain essential structural features such as order/disorder, interactions, and electrostatic potentials of their constituent domains. The results provide for the first time a dynamic view of a complete potyvirus CP, since experimental available structures so far obtained lack N- and C-terminal segments. The relevance of disorder in the most distal N-terminal subdomain, and the interaction of the less distal N-terminal subdomain with the highly ordered CP core, stand out as crucial characteristic for a viable CP. Preserving them proved of outmost importance to obtain viable potyviral CPs presenting peptides at their N-terminus.
Subject(s)
Potyvirus , Potyvirus/metabolism , Peptides , Capsid Proteins/metabolismABSTRACT
The wealth of variability amongst genes controlling immunity against potyviruses in pepper (Capsicum spp.) has been instrumental in understanding plant-virus co-evolution and major determinants of plant resistance durability. Characterization of the eukaryotic initiation factor 4E1 (eIF4E1), involved in mRNA translation, as the basis of potyvirus resistance in pepper initiated a large body of work that showed that recessive resistance to potyviruses and other single-stranded positive-sense RNA viruses resulted from mutations in eukaryotic initiation factors in many plant crop species. Combining mutations in different eIF4Es in the same pepper genotype had complex effects on the breadth of the resistance spectrum and on resistance durability, revealing a trade-off between these two traits. In addition, combining eIF4E1 mutations with a quantitatively resistant genetic background had a strong positive effect on resistance durability. Analysing the evolutionary forces imposed by pepper genotypes onto virus populations allowed identifying three key factors improving plant resistance durability: the complexity of mutational pathways involved in virus adaptation to the plant resistance, the decrease of competitivity induced by these mutations on the virus and the intensity of genetic drift imposed by plant genotypes on the virus during its infection cycle.
Subject(s)
Potyvirus , Potyvirus/genetics , Potyvirus/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Mutation , Plants , GenotypeABSTRACT
Regulation of protein synthesis is a strong determinant of potyviral pathogenicity. The Potyviridae family is the largest family of plant-infecting positive sense RNA viruses. Similar to the animal-infecting Picornaviridae family, the potyviral RNA genome lacks a 5' cap, and instead has a viral protein (VPg) linked to its 5' end. Potyviral genomes are mainly translated into one large polyprotein relying on a single translation event to express all their protein repertoire. In the absence of the 5' cap, the Potyviridae family depends on cis-acting elements in their 5' untranslated regions (UTR) to recruit the translation machinery. In this review, we summarize the diverse 5'UTR-driven, cap-independent translation mechanisms employed by the Potyviridae family including scanning-dependent mechanism, internal initiation, and the stimulatory role of the VPg. These mechanisms have direct implications on potyviral pathogenicity, including host range specificity and resistance. Finally, we discuss how these viral strategies could not only inform new avenues for engineering and/or breeding for crop resistance but would also provide opportunities for the development of biotechnological tools for large-scale protein production in plant systems.
Subject(s)
Potyviridae , Potyvirus , Animals , Potyvirus/genetics , Potyvirus/metabolism , Protein Biosynthesis , Plant Breeding , RNA/metabolism , Potyviridae/genetics , Potyviridae/metabolism , Plants/geneticsABSTRACT
Virus infection can increase drought tolerance of infected plants compared with noninfected plants; however, the mechanisms mediating virus-induced drought tolerance remain unclear. In this study, we demonstrate turnip mosaic virus (TuMV) infection increases Arabidopsis thaliana survival under drought compared with uninfected plants. To determine if specific TuMV proteins mediate drought tolerance, we cloned the coding sequence for each of the major viral proteins and generated transgenic A. thaliana that constitutively express each protein. Three TuMV proteins, 6K1, 6K2, and NIa-Pro, enhanced drought tolerance of A. thaliana when expressed constitutively in plants compared with controls. While in the control plant, transcripts related to abscisic acid (ABA) biosynthesis and ABA levels were induced under drought, there were no changes in ABA or related transcripts in plants expressing 6K2 under drought compared with well-watered conditions. Expression of 6K2 also conveyed drought tolerance in another host plant, Nicotiana benthamiana, when expressed using a virus overexpression construct. In contrast to ABA, 6K2 expression enhanced salicylic acid (SA) accumulation in both Arabidopsis and N. benthamiana. These results suggest 6K2-induced drought tolerance is mediated through increased SA levels and SA-dependent induction of plant secondary metabolites, osmolytes, and antioxidants that convey drought tolerance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Potyvirus , Arabidopsis/metabolism , Droughts , Potyvirus/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Plants, Genetically Modified , Abscisic Acid/metabolismABSTRACT
Coat proteins (CP) of the potato virus A virions (PVA) contain partially disordered N-terminal domains, which are necessary for performing vital functions of the virus. Comparative analysis of the structures of coat proteins (CPs) in the intact PVA virions and in the virus particles lacking N-terminal 32 amino acids (PVAΔ32) was carried out in this work based on the tritium planigraphy data. Using atomic-resolution structure of the potato virus Y potyvirus (PVY) protein, which is a homolog of the CP PVA, the available CP surfaces in the PVY virion were calculated and the areas of intersubunit/interhelix contacts were determined. For this purpose, the approach of Lee and Richards [Lee, B., and Richards, F. M. (1971) J. Mol. Biol., 55, 379-400] was used. Comparison of incorporation profiles of the tritium label in the intact and trypsin-degraded PVAΔ32 revealed position of the ΔN-peptide shielding the surface domain (a.a. 66-73, 141-146) and the interhelix zone (a.a. 161-175) of the PVA CP. Presence of the channels/cavities was found in the virion, which turned out to be partially permeable to tritium atoms. Upon removal of the ΔN-peptide, decrease in the label incorporation within the virion (a.a. 184-200) was also observed, indicating possible structural transition leading to the virion compactization. Based on the obtained data, we can conclude that part of the surface ΔN-peptide is inserted between the coils of the virion helix thus increasing the helix pitch and providing greater flexibility of the virion, which is important for intercellular transport of the viruses in the plants.
Subject(s)
Capsid Proteins , Potyvirus , Capsid Proteins/metabolism , Tritium/analysis , Tritium/metabolism , Proteolysis , Computer Simulation , Potyvirus/metabolism , Virion/metabolism , Peptides/metabolismABSTRACT
Potyviruses comprise the largest and most important group of plant positive-strand RNA viruses. The potyviral cell-to-cell movement protein P3N-PIPO is expressed via transcriptional slippage at a conserved GAAAAAA sequence, leading to insertion of an extra 'A' in a proportion of viral transcripts. Transcriptional slippage is determined by the potyviral replicase, the conserved slippery site, and its flanking nucleotides. Here, we investigate the dynamics of transcriptional slippage at different slip-site sequences, infection stages, and environmental conditions. We detect a modest increase in the level of transcripts with insertion towards later timepoints. In addition, we investigate the fate of transcripts with insertion by separately looking at different RNA subpopulations: (+)RNA, (-)RNA, translated RNA, and virion RNA. We find differences in insertional slippage between (+)RNA and (-)RNA but not other subpopulations. Our results suggest that there can be selection against the use of (-)RNAs with insertions as templates for transcription or replication and demonstrate that insertional slippage can occur at high frequency also during (-)RNA synthesis. Since transcripts with insertions are potential targets for degradation, we investigate the connection to nonsense-mediated decay (NMD). We find that these transcripts are targeted to NMD, but we only observe an impact on the level of transcripts with insertion when the insertional slippage rate is high. Together, these results further our understanding of the mechanism and elucidate the dynamics of potyviral transcriptional slippage. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Subject(s)
Potyvirus , Viral Proteins , Nucleotides/metabolism , Potyvirus/genetics , Potyvirus/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Nicotiana/genetics , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The development of recessive resistance by loss of susceptibility is a consistent strategy to combat and limit damages caused by plant viruses. Susceptibility genes can be turned into resistances, a feat that can either be selected among the plant's natural diversity or engineered by biotechnology. Here, we summarize the current knowledge on the phosphoglycerate kinases (PGK), which have emerged as a new class of susceptibility factors to single-stranded positive RNA viruses, including potyviruses. PGKs are metabolic enzymes involved in glycolysis and the carbon reduction cycle, encoded by small multigene families in plants. To fulfil their role in the chloroplast and in the cytosol, PGKs genes encode differentially addressed proteins. Here, we assess the diversity and homology of chloroplastic and cytosolic PGKs sequences in several crops and review the current knowledge on their redundancies during plant development, taking Arabidopsis as a model. We also show how PGKs have been shown to be involved in susceptibility-and resistance-to viruses. Based on this knowledge, and drawing from the experience with the well-characterized translation initiation factors eIF4E, we discuss how PGKs genes, in light of their subcellular localization, function in metabolism, and susceptibility to viruses, could be turned into efficient genetic resistances using genome editing techniques.
Subject(s)
Arabidopsis , Potyvirus , Arabidopsis/genetics , Chloroplasts/genetics , Eukaryotic Initiation Factor-4E/metabolism , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Potyvirus/genetics , Potyvirus/metabolismABSTRACT
Potyviral genomes encode just 11 major proteins and multifunctionality is associated with most of these proteins at different stages of the virus infection cycle. Some potyviral proteins modulate phytohormones and protein degradation pathways and have either pro- or anti-viral/insect vector functions. Our previous work demonstrated that the potyviral protein 6K1 has an antagonistic effect on vectors when expressed transiently in host plants, suggesting plant defenses are regulated. However, to our knowledge the mechanisms of how 6K1 alters plant defenses and how 6K1 functions are regulated are still limited. Here we show that the 6K1 from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and cysteine protease inhibitors when expressed in Nicotiana benthamiana relative to controls. 6K1 stability increased when cysteine protease activity was inhibited chemically, showing a mechanism to the rapid turnover of 6K1 when expressed in trans. Using RNAseq, qRT-PCR, and enzymatic assays, we demonstrate TuMV reprograms plant protein degradation pathways on the transcriptional level and increases 6K1 stability at later stages in the infection process. Moreover, we show 6K1 decreases plant protease activity in infected plants and increases TuMV accumulation in systemic leaves compared to controls. These results suggest 6K1 has a pro-viral function in addition to the anti-insect vector function we observed previously. Although the host targets of 6K1 and the impacts of 6K1-induced changes in protease activity on insect vectors are still unknown, this study enhances our understanding of the complex interactions occurring between plants, potyviruses, and vectors.
Subject(s)
Arabidopsis , Potyvirus , Peptide Hydrolases/metabolism , Plant Diseases , Potyvirus/metabolism , Proteolysis , Nicotiana , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2ß), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2ß. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2ß. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2ß. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic-tracking styryl-stained endosomes. The mutations were then introduced into a full-length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2ß for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.
Subject(s)
Potyvirus , Amino Acid Motifs , Endosomes/metabolism , Potyvirus/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virus ReplicationABSTRACT
The role of programmed cell death (PCD) in hypersensitive response (HR)-conferred resistance depends on the type of host-pathogen interaction and therefore has to be studied for each individual pathosystem. Here we present and explain the protocol for studying the role of PCD in HR-conferred resistance in potato plants in the interaction with the viral pathogen. As an experimental system, we use genotype Rywal, where the virus spread is restricted and HR PCD develops 3 days post potato virus Y (PVY) inoculation. As a control of virus multiplication and spread, we include its transgenic counterpart impaired in salicylic acid (SA) accumulation (NahG-Rywal), in which the HR-PCD occurs but the spread of the virus is not restricted. To follow the occurrence of virus-infected cells and/or virus multiplication outside the cell death zone, we use GFP-tagged PVY (PVY-N605(123)-GFP) which can be monitored by confocal microscopy. Any other plant-pathogen system which results in PCD development could be studied using a modified version of this protocol.
Subject(s)
Potyvirus , Solanum tuberosum , Cell Death , DNA Viruses , Microscopy, Confocal , Plant Diseases/genetics , Potyvirus/genetics , Potyvirus/metabolism , Salicylic Acid/metabolism , Solanum tuberosum/geneticsABSTRACT
Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1-RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1NIa . StPBS1NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1NIa .
Subject(s)
Arabidopsis , Potyvirus , Solanum tuberosum , Virus Diseases , Peptide Hydrolases/metabolism , Plant Diseases , Potyvirus/metabolismABSTRACT
Turnip mosaic virus is a devastating potyvirus infecting many economically important brassica crops. In response to this, the plant host engages its RNA silencing machinery, involving AGO proteins, as a prominent strategy to restrain turnip mosaic virus (TuMV) infection. It has also been shown that the mRNA decay components DCP2 and VCS partake in viral infection suppression. Here, we report that the mRNA decapping components LSM1, PAT1, PATH1, and PATH2 are essential for TuMV infection. More specifically, lsm1a/lsm1b double mutants and pat1/path1/path2 triple mutants in summ2 background exhibit resistance to TuMV. Concurrently, we observed that TuMV interferes with the decapping function of LSM1 and PAT proteins as the mRNA-decay target genes UGT87A2 and ASL9 accumulate during TuMV infection. Moreover, as TuMV coat protein can be specifically found in complexes with PAT proteins but not LSM1, this suggests that TuMV "hijacks" decapping components via PAT proteins to support viral infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Potyvirus , Plant Diseases , Potyvirus/genetics , Potyvirus/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mottle virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.
Subject(s)
Chloroplast Proteins/metabolism , Cyclopentanes/metabolism , Intramolecular Oxidoreductases/metabolism , Oxylipins/metabolism , Plant Diseases/virology , Potyvirus/metabolism , Thylakoids/metabolism , Host-Parasite Interactions/physiology , Plant Growth Regulators/metabolism , Plant Immunity , Virus Diseases/virologyABSTRACT
Binding of phosphorylated eIFiso4E with viral genome-linked protein (VPg) of turnip mosaic virus was examined by stopped-flow, fluorescence, circular dichroism (CD) spectroscopy, and molecular docking analysis. Phosphorylation of eIFiso4E increased (4-fold) the binding rates as compared to unphosphorylated eIFiso4E with VPg. Stopped-flow kinetic studies of phosphorylated eIFiso4E with VPg showed a concentration-independent conformational change. The dissociation rate was about 3-fold slower for eIFiso4EâVPg complex upon phosphorylation. Phosphorylation enhanced the association rates and lowered the dissociation rates for the eIFiso4EâVPg binding, with having higher preferential binding to eIFiso4Ep. Binding rates for the interaction of eIFiso4Ep with VPg increased (6-fold) with an increase in temperature, 278 K to 298 K. The activation energies for binding of eIFiso4Ep and eIFiso4E with VPg were 37.2 ± 2.8 and 52.6 ± 3.6 kJ/mol, respectively. Phosphorylation decreased the activation energy for the binding of eIFiso4E to VPg. The reduced energy barrier suggests more stable platform for eIFiso4EpâVPg initiation complex formation, which was further supported by molecular docking analysis. Moreover, far-UV CD studies revealed that VPg formed complex with eIFiso4Ep with substantial change in the secondary structure. These results suggested that phosphorylation, not only reduced the energy barrier and dissociation rate but also enhanced binding rate, and an overall conformational change, which provides a more stable platform for efficient viral translation.