Search Results (17)

Viral infections and their vector dynamics pose a major threat to potatoes (Solanum tuberosum L.) worldwide, urgently needing an integrated understanding of the molecular and ecological interactions in this tripartite system. This review describes the major potato viruses, namely potato virus Y (PVY), the potato leafroll virus (PLRV), and potato virus X (PVX), with an emphasis on their infection and replication strategies in plants, as well as their movement within them. It also discusses plant responses to these viruses by uncovering RNA silencing, resistance (R) genes, and hormonal signaling. The complex dynamics of virus–vector interactions are discussed, considering the modes of transmission-persistent, non-persistent and semi-persistent—the role of viral proteins such as HC-Pro in determining vector specificity and adaptations in vectors that facilitate virus dissemination. This article discusses how vectors select potato plants, with an emphasis on the role played by plant-excreted volatiles and vector-applied saliva in plant defense. It also discusses host genes that contribute to vector resistance. This review provides an overview of the interactions between potato plants, viruses, and vectors and shows how viruses influence plant–vector interactions, the molecular pathways shared, and the altered gene expression profiles due to these interactions. The review offers an integrated perspective essential for developing sustainable and precise control strategies against potato viral pathogens under changing climatic conditions. Full article

Phytophthora sojae is a type of pathogenic oomycete that causes Phytophthora root stem rot (PRSR), which can seriously affect the soybean yield and quality. To subvert immunity, P. sojae secretes a large quantity of effectors. However, the molecular mechanisms regulated by most P. sojae effectors, and their host targets remain unexplored. Previous studies have shown that the expression of PsAvh113, an effector secreted by Phytophthora sojae, enhances viral RNA accumulations and symptoms in Nicotiana benthamiana via VIVE assay. In this study, we analyzed RNA-sequencing data based on disease symptoms in N. benthamiana leaves that were either mocked or infiltrated with PVX carrying the empty vector (EV) and PsAvh113. We identified 1769 differentially expressed genes (DEGs) dependent on PsAvh113. Using stricter criteria screening and Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis of DEGs, we found that 38 genes were closely enriched in response to PsAvh113 expression. We selected three genes of N. benthamiana (NbNAC86, NbMyb4, and NbERF114) and found their transcriptional levels significantly upregulated in N. benthamiana infected with PVX carrying PsAvh113. Furthermore, individual silencing of these three genes promoted P. capsici infection, while their overexpression increased resistance to P. capsici in N. benthamiana. Our results show that PsAvh113 interacts with transcription factors NbMyb4 and NbERF114 in vivo. Collectively, these data may help us understand the pathogenic mechanism of effectors and manage PRSR in soybeans. Full article

Faba bean is an important cool-season edible legume crop that is constantly threatened by abiotic stresses such as drought. The basic leucine zipper (bZIP) gene family is one of the most abundant and diverse families of transcription factors in plants. It regulates plant growth and development and plays an important role in the response to biotic and abiotic stresses. In this study, we identified 18 members of the faba bean bZIP transcription factor family at the genome-wide level based on previous faba bean drought stress transcriptome sequencing data. A phylogenetic tree was constructed to group the 18 VfbZIP proteins into eight clades. Analysis of cis-acting elements in the promoter region suggested that these 18 VfbZIPs may be involved in regulating abiotic stress responses such as drought. Transcriptome data showed high expression of seven genes (VfbZIP1, VfbZIP2, VfbZIP5, VfbZIP7, VfbZIP15, VfbZIP17, and VfbZIP18) in the drought-tolerant cultivar under drought stress, in which VfbZIP1, VfbZIP2, and VfbZIP5 were consistently expressed as detected by quantitative real-time polymerase chain reaction (qRT-PCR) compared to the transcriptome data. Ectopic overexpression of the three VfbZIPs in tobacco, based on the potato Virus X (PVX) vector, revealed that VfbZIP5 enhanced the drought tolerance. Overexpressed VfbZIP5 in plants showed lower levels of proline (PRO), malondialdehyde (MDA), and peroxidase (POD) compared to those overexpressing an empty vector under 10 days of drought stress. Protein-protein interaction (PPI) analysis showed that VfbZIP5 interacted with seven proteins in faba bean, including VfbZIP7 and VfbZIP10. The results depict the importance of VfbZIPs in response to drought stress, and they would be useful for the improvement of drought tolerance. Full article

Sugarcane yellow leaf virus (SCYLV), a member of the genus Polerovirus in the family Luteoviridae, causes severe damage and represents a great threat to sugarcane cultivation and sugar industry development. In this study, inoculation of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the SCYLV P0 gene induced typical mosaic, leaf rolling symptoms and was associated with a hypersensitive-like response (HLR) necrosis symptom, which is accompanied with a systemic burst of H₂O₂ and also leads to higher PVX viral genome accumulation levels. Our results demonstrate that SCYLV P0 is a pathogenicity determinant and plays important roles in disease development. To further explore its function in pathogenic processes, a yeast two-hybrid assay was performed to screen the putative P0-interacting host factors. The recombinant plasmid pGBKT7-P0 was constructed as a bait and transformed into the yeast strain Y2HGold. The ROC22 cultivar (an important parental resource of the main cultivar in China) cDNA prey library was constructed and screened by co-transformation with the P0 bait. We identified 28 potential interacting partners including those involved in the optical signal path, plant growth and development, transcriptional regulation, host defense response, and viral replication. To our knowledge, this is the first time we have reported the host proteins interacting with the P0 virulence factor encoded by sugarcane yellow leaf virus. This study not only provides valuable insights into elucidating the molecular mechanism of the pathogenicity of SCYLV, but also sheds light on revealing the probable new pathogenesis of Polerovirus in the future. Full article

With the increasing global population, saving crops from diseases caused by different kinds of bacteria, fungi, viruses, and nematodes is essential. Potato is affected by various diseases, destroying many crops in the field and storage. In this study, we developed potato lines resistant to fungi and viruses, Potato Virus X (PVX) and Potato Virus Y (PVY), by inoculating chitinase for fungi and shRNA designed against the mRNA of the coat protein of PVX and PVY, respectively. The construct was developed using the pCAMBIA2301 vector and transformed into AGB-R (red skin) potato cultivar using Agrobacterium tumefaciens. The crude protein extract of the transgenic potato plant inhibited the growth of Fusarium oxysporum from ~13 to 63%. The detached leaf assay of the transgenic line (SP-21) showed decreased necrotic spots compared to the non-transgenic control when challenged with Fusarium oxysporum. The transgenic line, SP-21, showed maximum knockdown when challenged with PVX and PVY, i.e., 89 and 86%, while transgenic line SP-148 showed 68 and 70% knockdown in the PVX- and PVY-challenged conditions, respectively. It is concluded from this study that the developed transgenic potato cultivar AGB-R showed resistance against fungi and viruses (PVX and PVY). Full article

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting 0.5% to 1% of the population. It could ultimately result in joint destruction, functional decline, work disability, and enhanced mortality. Cyclic citrullinated peptide antibodies (CCP Abs) are useful biomarkers for the early detection and diagnosis of RA. In this study, we used plant viral-based expression vectors that produce rapidly large quantities of CCP-specific monoclonal antibodies. Heavy and light chain genes of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant viral-based expression vectors, TMV and PVX. A cyclic citrullinated peptide monoclonal antibody was produced in Nicotiana benthamiana through an Agrobacterium-mediated transient expression system. The expression of CCP mAb in tobacco plants was confirmed by dot blot, western blot analysis, and enzyme-linked immunosorbent assays (ELISA). It was shown that tobacco plants could accumulate CCP mAbs up to 0.35% of total soluble protein. Accumulated CCP mAb from infiltrated leaves was purified by protein G affinity chromatography. Immunoblot assays and ELISA showed plant-produced CCP mAbs successfully bound to a synthetic CCP peptide antigen. This system provides a fast strategy for the production of pharmaceutical CCP mAbs in tobacco plants. Full article

Phytoplasmas are uncultivable, phloem-limited, phytopathogenic bacteria that represent a major threat to agriculture worldwide. Phytoplasma membrane proteins are in direct contact with hosts and presumably play a crucial role in phytoplasma spread within the plant as well as by the insect vector. Three highly abundant types of immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). Although recent results indicate that Amp is involved in host specificity by interacting with host proteins such as actin, little is known about the pathogenicity of IDP in plants. In this study, we identified an antigenic membrane protein (Amp) of rice orange leaf phytoplasma (ROLP), which interacts with the actin of its vector. In addition, we generated Amp-transgenic lines of rice and expressed Amp in tobacco leaves by the potato virus X (PVX) expression system. Our results showed that the Amp of ROLP can induce the accumulation of ROLP and PVX in rice and tobacco plants, respectively. Although several studies have reported interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins, this example demonstrates that Amp protein can not only interact with the actin protein of its insect vector but can also directly inhibit host defense responses to promote the infection. The function of ROLP Amp provides new insights into the phytoplasma-host interaction. Full article

Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes’ presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300–400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80–100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen. Full article

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement. Full article

Similarly to other potyvirids, the bymovirus wheat yellow mosaic virus (WYMV) encodes a P3N-PIPO protein that is expressed by frameshifting occurring within the open reading frame of the P3 protein. P3N-PIPO is known to be essential for the cell-to-cell movement of several potyviruses, but this has not yet been confirmed for the WYMV. Here, we show that the WYMV P3N-PIPO protein influences disease symptom formation. Infection of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the WYMV P3N-PIPO gene induced more severe disease symptoms and resulted in higher virus accumulation levels than did infection with PVX lacking the P3N-PIPO gene. N. benthamiana P3N-PIPO-interacting proteins were identified through co-immunoprecipitation (Co-IP) coupled with LC-MS/MS (mass spectrometry), and the interaction between P3N-PIPO and the N. benthamiana receptor-like kinase NbRLK6 was further verified by Co-IP and bimolecular fluorescence complementation (BiFC) of transiently-expressed proteins. Furthermore, our investigation showed that the disease symptom severity and accumulation level of PVX-P3N-PIPO were decreased in N. benthamiana plants when NbRLK6 expression was reduced by tobacco rattle virus-induced gene silencing. Full article

We have developed a Potato virus X (PVX)-based vector system compatible with the GoldenBraid 2.0 (GB) cloning strategy to transiently express heterologous proteins or peptides in plants for biotechnological purposes. This vector system consists of three domestication vectors carrying three GB parts—the cauliflower mosaic virus (CaMV) 35S promoter with PVX upstream of the second subgenomic promoter of the PVX coat protein (PVX CP SGP), nopaline synthase (NOS) terminator with PVX downstream of the first PVX CP SGP and the gene of interest (GOI). The full-length PVX clone carrying the sequence encoding a green fluorescent protein (GFP) as GOI was incorporated into the binary GB vector in a one-step reaction of three GB parts using the four-nucleotide GB standard syntax. We investigated whether the obtained vector named GFP/pGBX enables systemic PVX infection and expression of GFP in Nicotiana benthamiana plants. We show that this GB-compatible vector system can be used for simple and efficient assembly of PVX-based expression constructs and that it meets the current need for interchange of standard biological parts used in different expression systems. Full article

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems. Full article

Plant-based nanotechnology programs using virus-like particles (VLPs) and virus nanoparticles (VNPs) are emerging platforms that are increasingly used for a variety of applications in biotechnology and medicine. Tobacco mosaic virus (TMV) and potato virus X (PVX), by virtue of having high aspect ratios, make ideal platforms for drug delivery. TMV and PVX both possess rod-shaped structures and single-stranded RNA genomes encapsidated by their respective capsid proteins and have shown great promise as drug delivery systems. Cowpea mosaic virus (CPMV) has an icosahedral structure, and thus brings unique benefits as a nanoparticle. The uses of these three plant viruses as either nanostructures or expression vectors for high value pharmaceutical proteins such as vaccines and antibodies are discussed extensively in the following review. In addition, the potential uses of geminiviruses in medical biotechnology are explored. The uses of these expression vectors in plant biotechnology applications are also discussed. Finally, in this review, we project future prospects for plant viruses in the fields of medicine, human health, prophylaxis, and therapy of human diseases. Full article

P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G₂A₆ slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G₂A₆ site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G₂A₆ site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana. Full article

‘Candidatus Liberibacter asiaticus’ (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H₂O₂ accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants. Full article