RESUMEN

Rubisco small subunit (RbcS), a core component with crucial effects on the structure and kinetic properties of the Rubisco enzyme, plays an important role in response to plant growth, development, and various stresses. Although Rbcs genes have been characterized in many plants, their muti-functions in soybeans remain elusive. In this study, a total of 11 GmRbcS genes were identified and subsequently divided into three subgroups based on a phylogenetic relationship. The evolutionary analysis revealed that whole-genome duplication has a profound effect on GmRbcSs. The cis-acting elements responsive to plant hormones, development, and stress-related were widely found in the promoter region. Expression patterns based on the RT-qPCR assay exhibited that GmRbcS genes are expressed in multiple tissues, and notably Glyma.19G046600 (GmRbcS8) exhibited the highest expression level compared to other members, especially in leaves. Moreover, differential expressions of GmRbcS genes were found to be significantly regulated by exogenous plant hormones, demonstrating their potential functions in diverse biology processes. Finally, the function of GmRbcS8 in enhancing soybean resistance to soybean mosaic virus (SMV) was further determined through the virus-induced gene silencing (VIGS) assay. All these findings establish a strong basis for further elucidating the biological functions of RbcS genes in soybeans.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Glycine max , Filogenia , Reguladores del Crecimiento de las Plantas , Potyvirus , Glycine max/genética , Glycine max/virología , Glycine max/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Potyvirus/patogenicidad , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Perfilación de la Expresión Génica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Genoma de Planta , Estudio de Asociación del Genoma Completo , Resistencia a la Enfermedad/genética , Familia de Multigenes

RESUMEN

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

Asunto(s)

Carica , Enfermedades de las Plantas , Potyvirus , Carica/virología , Carica/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Potyvirus/genética , Potyvirus/patogenicidad , Plantas Modificadas Genéticamente/genética , Fitomejoramiento/métodos , Resistencia a la Enfermedad/genética , Edición Génica/métodos , Proteínas de la Cápside/genética , Silenciador del Gen

RESUMEN

East Asian Passiflora virus (EAPV) causes passionfruit woodiness disease, a major threat limiting passionfruit production in eastern Asia, including Taiwan and Vietnam. In this study, an infectious cDNA clone of a Taiwanese severe isolate EAPV-TW was tagged with a green fluorescent protein (GFP) reporter to monitor the virus in plants. Nicotiana benthamiana and yellow passionfruit plants inoculated with the construct showed typical symptoms of EAPV-TW. Based on our previous studies on pathogenicity determinants of potyviral HC-Pros, a deletion of six amino acids (d6) alone and its association with a point mutation (F8I, simplified as I8) were conducted in the N-terminal region of the HC-Pro gene of EAPV-TW to generate mutants of EAPV-d6 and EAPV-d6I8, respectively. The mutant EAPV-d6I8 caused infection without conspicuous symptoms in N. benthamiana and yellow passionfruit plants, while EAPV-d6 still induced slight leaf mottling. EAPV-d6I8 was stable after six passages under greenhouse conditions and displayed a zigzag pattern of virus accumulation, typical of a beneficial protective virus. The cross-protection effectiveness of EAPV-d6I8 was evaluated in both N. benthamiana and yellow passionfruit plants under greenhouse conditions. EAPV-d6I8 conferred complete cross-protection (100%) against the wild-type EAPV-TW-GFP in both N. benthamiana and yellow passionfruit plants, as verified by no severe symptoms, no fluorescent signals, and PCR-negative status for GFP. Furthermore, EAPV-d6I8 also provided complete protection against Vietnam's severe strain EAPV-GL1 in yellow passionfruit plants. Our results indicate that the attenuated mutant EAPV-d6I8 has great potential to control EAPV in Taiwan and Vietnam via cross-protection.

Asunto(s)

Mutación , Enfermedades de las Plantas , Potyvirus , Proteínas Virales , Protección Cruzada , Cisteína Endopeptidasas , Nicotiana/virología , Nicotiana/genética , Passiflora/virología , Passiflora/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/prevención & control , Potyvirus/genética , Eliminación de Secuencia , Taiwán , Vietnam , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

The eukaryotic mRNA surveillance pathway, a pivotal guardian of mRNA fidelity, stands at the nexus of diverse biological processes, including antiviral immunity. Despite the recognized function of splicing factors on mRNA fate, the intricate interplay shaping the mRNA surveillance pathway remains elusive. We illustrate that the conserved splicing factor U2 snRNP auxiliary factor large subunit B (U2AF65B) modulates splicing of mRNA surveillance complex, contributing to transcriptomic homeostasis in maize. The functionality of the mRNA surveillance pathway requires ZmU2AF65B-mediated normal splicing of upstream frameshift 3 (ZmUPF3) pre-mRNA, encoding a core factor in this pathway. Intriguingly, sugarcane mosaic virus (SCMV)-coded nuclear inclusion protein a protease (NIa-Pro) hinders the splicing function of ZmU2AF65B. Furthermore, NIa-Pro disrupts ZmU2AF65B binding to ZmUPF3 pre-mRNA, leading to dysregulated splicing of ZmUPF3 transcripts and, consequently, impairing mRNA surveillance, thus facilitating viral infection. Together, this study establishes that splicing governs the mRNA surveillance pathway and identifies a pathogenic protein capable of disrupting this regulation to compromise RNA immunity.

Asunto(s)

Potyvirus , Empalme del ARN , ARN Mensajero , Zea mays , Zea mays/virología , Zea mays/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Potyvirus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Factor de Empalme U2AF/metabolismo , Factor de Empalme U2AF/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Regulación de la Expresión Génica de las Plantas

RESUMEN

KEY MESSAGE: The Ra extreme resistance against potato virus A was mapped to the upper of chromosome 4 in tetraploid potato. Potato virus A (PVA) is one of the major viruses affecting potato worldwide and can cause serious disease symptoms and yield losses. Previously, we determined that potato cultivar Barbara harbors Rysto (genotype: Ryryryry) and Ra (genotype: Rararara) that each independently confer extreme resistance to PVA. In this study, employing a combination of next-generation sequencing and bulked-segregant analysis, we further located this novel Ra on chromosome 4 using a tetraploid BC1 potato population derived from a Ry-free progeny (Rararararyryryry) of Barbara (RarararaRyryryry) × F58050 (rararararyryryry). Using 29 insertion-deletion (InDel) markers spanning chromosome 4, Ra was delimited by the InDel markers M8-83 and M10-8 within a genetic interval of 1.46 cM, corresponding to a 1.86-Mb genomic region in the potato DM reference genome. The InDel marker M10-8, which is closely linked with the resistance against PVA in the Ry-free segregating populations, was then used to screen 43 selected Rysto-free tetraploid potato breeding clones. The phenotype to PVA was significantly correlated with the present/absent of the marker, albeit with a 9.3% false positive rate and a 14.0% false negative rate. These findings are of importance in furthering the cloning of Ra and employing the marker-assisted selection for PVA resistance.

Asunto(s)

Mapeo Cromosómico , Resistencia a la Enfermedad , Enfermedades de las Plantas , Potyvirus , Solanum tuberosum , Solanum tuberosum/genética , Solanum tuberosum/virología , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Potyvirus/patogenicidad , Fenotipo , Genotipo , Marcadores Genéticos , Mutación INDEL , Cromosomas de las Plantas/genética , Tetraploidía , Fitomejoramiento

RESUMEN

Ethylene response factors (ERFs) have been associated with biotic stress in Arabidopsis, while their function in non-model plants is still poorly understood. Here we investigated the role of potato ERF StPti5 in plant immunity. We show that StPti5 acts as a susceptibility factor. It negatively regulates potato immunity against potato virus Y and Ralstonia solanacearum, pathogens with completely different modes of action, and thereby has a different role than its orthologue in tomato. Remarkably, StPti5 is destabilised in healthy plants via the autophagy pathway and accumulates exclusively in the nucleus upon infection. We demonstrate that StEIN3 and StEIL1 directly bind the StPti5 promoter and activate its expression, while synergistic activity of the ethylene and salicylic acid pathways is required for regulated StPti expression. To gain further insight into the mode of StPti5 action in attenuating potato defence responses, we investigated transcriptional changes in salicylic acid deficient potato lines with silenced StPti5 expression. We show that StPti5 regulates the expression of other ERFs and downregulates the ubiquitin-proteasome pathway as well as several proteases involved in directed proteolysis. This study adds a novel element to the complex puzzle of immune regulation, by deciphering a two-level regulation of ERF transcription factor activity in response to pathogens.

Asunto(s)

Etilenos , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas , Inmunidad de la Planta , Proteínas de Plantas , Potyvirus , Regiones Promotoras Genéticas , Ralstonia solanacearum , Ácido Salicílico , Solanum tuberosum , Solanum tuberosum/microbiología , Solanum tuberosum/inmunología , Solanum tuberosum/genética , Solanum tuberosum/virología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Etilenos/metabolismo , Ralstonia solanacearum/fisiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Ácido Salicílico/metabolismo , Potyvirus/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica , Complejo de la Endopetidasa Proteasomal/metabolismo , Autofagia , Núcleo Celular/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas Portadoras , Comovirus , Nicotiana , Enfermedades de las Plantas , Proteínas de Plantas , Vigna , Comovirus/metabolismo , Comovirus/fisiología , Comovirus/genética , Vigna/virología , Vigna/metabolismo , Nicotiana/virología , Nicotiana/metabolismo , Nicotiana/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Enfermedades de las Plantas/virología , Proteasas de Cisteína/metabolismo , Proteasas de Cisteína/genética , Plantas Modificadas Genéticamente , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Potyvirus/fisiología , Potyvirus/metabolismo , Endopeptidasas

RESUMEN

Manipulating evolutionary forces imposed by hosts on pathogens like genetic drift and selection could avoid the emergence of virulent pathogens. For instance, increasing genetic drift could decrease the risk of pathogen adaptation through the random fixation of deleterious mutations or the elimination of favorable ones in the pathogen population. However, no experimental proof of this approach is available for a plant-pathogen system. We studied the impact of pepper (Capsicum annuum) lines carrying the same major resistance gene but contrasted genetic backgrounds on the evolution of Potato virus Y (PVY). The pepper lines were chosen for the contrasted levels of genetic drift (inversely related to Ne, the effective population size) they exert on PVY populations, as well as for their contrasted resistance efficiency (inversely related to the initial replicative fitness, Wi, of PVY in these lines). Experimental evolution was performed by serially passaging 64 PVY populations every month on six contrasted pepper lines during seven months. These PVY populations exhibited highly divergent evolutionary trajectories, ranging from viral extinctions to replicative fitness gains. The sequencing of the PVY VPg cistron, where adaptive mutations are likely to occur, allowed linking these replicative fitness gains to parallel adaptive nonsynonymous mutations. Evolutionary trajectories were well explained by the genetic drift imposed by the host. More specifically, Ne, Wi and their synergistic interaction played a major role in the fate of PVY populations. When Ne was low (i.e. strong genetic drift), the final PVY replicative fitness remained close to the initial replicative fitness, whereas when Ne was high (i.e. low genetic drift), the final PVY replicative fitness was high independently of the replicative fitness of the initially inoculated virus. We show that combining a high resistance efficiency (low Wi) and a strong genetic drift (low Ne) is the best solution to increase resistance durability, that is, to avoid virus adaptation on the long term.

Asunto(s)

Capsicum , Flujo Genético , Enfermedades de las Plantas , Potyvirus , Capsicum/virología , Capsicum/genética , Potyvirus/genética , Potyvirus/patogenicidad , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Interacciones Huésped-Patógeno/genética , Resistencia a la Enfermedad/genética , Adaptación Fisiológica/genética , Mutación

RESUMEN

Since the establishment of procedures for the safety assessment of food products that use recombinant DNA technology, the manufacture, import, and sale of genetically modified (GM) foods that have not undergone safety assessment are prohibited under the Food Sanitation Act. Therefore, a performance study to confirm the GM food testing operations of each laboratory is very important to ensure the reliability of the GM food monitoring system. In 2022, GM papaya line PRSV-YK-which has not yet been authorized in Japan-was selected for testing, and a papaya paste and a DNA solution were used as the test samples. With these samples, a laboratory performance study of the DNA extraction and real-time PCR operations was conducted. This confirmed that the 18 participating laboratories were generally performing the DNA extraction and real-time PCR operations correctly. However, some laboratories using certain DNA amplification reagent with some real-time PCR instruments were not able to determine the PRSV-YK detection test. This suggests that the PRSV-YK detection test may not be able to correctly detect samples containing GM papaya when performed with these combinations of instruments and reagent. In order to ensure the reliability of the PRSV-YK detection test, it is necessary to examine in detail how the combination of DNA polymerase reagents and real-time PCR instruments affects the detection limit, and to implement an appropriate solution.

Asunto(s)

Carica , Alimentos Modificados Genéticamente , Plantas Modificadas Genéticamente , Carica/genética , ADN de Plantas/genética , ADN de Plantas/análisis , Análisis de los Alimentos/métodos , Inocuidad de los Alimentos , Japón , Plantas Modificadas Genéticamente/genética , Potyvirus/genética , Potyvirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados

RESUMEN

In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.

Asunto(s)

Carica , ADN Polimerasa Dirigida por ADN , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Carica/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Plantas Modificadas Genéticamente/genética , Alimentos Modificados Genéticamente , Caulimovirus/genética , Potyvirus/genética , Potyvirus/aislamiento & purificación

RESUMEN

The interactions among plant viruses, insect vectors, and host plants have been well studied; however, the roles of insect viruses in this system have largely been neglected. We investigated the effects of MpnDV infection on aphid and PVY transmission using bioassays, RNA interference (RNAi), and GC-MS methods and green peach aphid (Myzus persicae (Sulzer)), potato virus Y (PVY), and densovirus (Myzus persicae nicotianae densovirus, MpnDV) as model systems. MpnDV increased the activities of its host, promoting population dispersal and leading to significant proliferation in tobacco plants by significantly enhancing the titer of the sesquiterpene (E)-ß-farnesene (EßF) via up-regulation of expression levels of the MpFPPS1 gene. The proliferation and dispersal of MpnDV-positive individuals were faster than that of MpnDV-negative individuals in PVY-infected tobacco plants, which promoted the transmission of PVY. These results combined showed that an insect virus may facilitate the transmission of a plant virus by enhancing the locomotor activity and population proliferation of insect vectors. These findings provide novel opportunities for controlling insect vectors and plant viruses, which can be used in the development of novel management strategies.

Asunto(s)

Áfidos , Densovirus , Nicotiana , Enfermedades de las Plantas , Áfidos/virología , Áfidos/fisiología , Animales , Nicotiana/virología , Nicotiana/parasitología , Enfermedades de las Plantas/virología , Densovirus/fisiología , Densovirus/genética , Potyvirus/fisiología , Potyvirus/patogenicidad , Sesquiterpenos/metabolismo , Virus de Plantas/fisiología , Virus de Plantas/patogenicidad

RESUMEN

Vertical transmission, the transfer of pathogens across generations, is a critical mechanism for the persistence of plant viruses. The transmission mechanisms are diverse, involving direct invasion through the suspensor and virus entry into developing gametes before achieving symplastic isolation. Despite the progress in understanding vertical virus transmission, the environmental factors influencing this process remain largely unexplored. We investigated the complex interplay between vertical transmission of plant viruses and pollination dynamics, focusing on common bean (Phaseolus vulgaris). The intricate relationship between plants and pollinators, especially bees, is essential for global ecosystems and crop productivity. We explored the impact of virus infection on seed transmission rates, with a particular emphasis on bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV). Under controlled growth conditions, BCMNV exhibited the highest seed transmission rate, followed by BCMV and CMV. Notably, in the field, bee-pollinated BCMV-infected plants showed a reduced transmission rate compared to self-pollinated plants. This highlights the influence of pollinators on virus transmission dynamics. The findings demonstrate the virus-specific nature of seed transmission and underscore the importance of considering environmental factors, such as pollination, in understanding and managing plant virus spread.

Asunto(s)

Phaseolus , Enfermedades de las Plantas , Polinización , Animales , Enfermedades de las Plantas/virología , Abejas/virología , Phaseolus/virología , Semillas/virología , Transmisión Vertical de Enfermedad Infecciosa , Cucumovirus/fisiología , Potyvirus/fisiología

RESUMEN

BACKGROUND: Soybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear. RESULTS: Here, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element. CONCLUSION: Our findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.

Asunto(s)

Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Glycine max , Enfermedades de las Plantas , Proteínas de Plantas , Potyvirus , Factores de Transcripción , Glycine max/virología , Glycine max/genética , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Potyvirus/fisiología , Potyvirus/patogenicidad , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas

RESUMEN

We had previously reported that a plum pox virus (PPV)-based chimera that had its P1-HCPro bi-cistron replaced by a modified one from potato virus Y (PVY) increased its virulence in some Nicotiana benthamiana plants, after mechanical passages. This correlated with the natural acquisition of amino acid substitutions in several proteins, including in HCPro at either position 352 (Ile→Thr) or 454 (Leu→Arg), or of mutations in non-coding regions. Thr in position 352 is not found among natural potyviruses, while Arg in 454 is a reversion to the native PVY HCPro amino acid. We show here that both mutations separately contributed to the increased virulence observed in the passaged chimeras that acquired them, and that Thr in position 352 is no intragenic suppressor to a Leu in position 454, because their combined effects were cumulative. We demonstrate that Arg in position 454 improved HCPro autocatalytic cleavage, while Thr in position 352 increased its accumulation and the silencing suppression of a reporter in agropatch assays. We assessed infection by four cloned chimera variants expressing HCPro with none of the two substitutions, one of them or both, in wild-type versus DCL2/4-silenced transgenic plants. We found that during infection, the transgenic context of altered small RNAs affected the accumulation of the four HCPro variants differently and hence, also infection virulence.

Asunto(s)

Sustitución de Aminoácidos , Nicotiana , Potyvirus , Proteínas Virales , Virulencia/genética , Nicotiana/virología , Potyvirus/patogenicidad , Potyvirus/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Enfermedades de las Plantas/virología , Quimera , Virus Eruptivo de la Ciruela/patogenicidad , Virus Eruptivo de la Ciruela/genética , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Mutación/genética

RESUMEN

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

Asunto(s)

Resistencia a la Enfermedad , MicroARNs , Nicotiana , Enfermedades de las Plantas , Plantas Modificadas Genéticamente , Potyvirus , MicroARNs/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/virología , Plantas Modificadas Genéticamente/inmunología , Nicotiana/genética , Nicotiana/virología , Nicotiana/inmunología , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Resistencia a la Enfermedad/genética , Potyvirus/patogenicidad , Potyvirus/genética , Interferencia de ARN , Glycine max/genética , Glycine max/virología , Glycine max/inmunología

RESUMEN

Potyvirus genomes are expressed as polyproteins that are autocatalytically cleaved to produce 10 to 12 multifunctional proteins, among which P1 is the most variable. It has long been hypothesized that P1 plays role(s) in host adaptation and host specificity. We tested this hypothesis using two phylogenetically distinct potyviruses: soybean mosaic virus (SMV), with a narrow host range, and clover yellow vein virus (ClYVV), with a broader host range. When the full-length P1 cistron of SMV-N was replaced with P1 from ClYVV-No.30, the chimera systemically infected only SMV-N-permissive hosts. Hence, there were no changes in the host range or host specificity of the chimeric viruses. Despite sharing only 20.3% amino acid sequence identity, predicted molecular models of P1 proteins from SMV-N and ClYVV-No.30 showed analogous topologies. These observations suggest that P1 of ClYVV-No.30 can functionally replace P1 of SMV-N. However, the P1 proteins of these two potyviruses are not determinants of host specificity and host range.

Asunto(s)

Especificidad del Huésped , Enfermedades de las Plantas , Potyvirus , Proteínas Virales , Potyvirus/genética , Potyvirus/fisiología , Enfermedades de las Plantas/virología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Glycine max/virología , Nicotiana/virología , Filogenia

RESUMEN

The Papaya ringspot virus (PRSV)-resistant genetically modified (GM) papaya 'Huanong No.1' has been certified as safe for consumption and widely planted in China for about 18 years. To protect consumers' rights and facilitate government supervision and monitoring, it is necessary to establish a simple, rapid, and specific detection method for 'Huanong No.1'. Herein, we developed a platform based on recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a for the detection of 'Huanong No.1'. The RPA-CRISPR-Cas12a platform was found to have high specificity, with amplification signals only present in 'Huanong No.1'. Additionally, the platform was highly sensitive, with a limit of detection (LOD) of approximately 20 copies. The detection process was fast and could be completed in less than 1 h. This novel platform enables the rapid on-site visualization detection of 'Huanong No.1', eliminating dependence on laboratory conditions and specialized instruments, and can serve as a technical reference for the rapid detection of other GM plants.

Asunto(s)

Sistemas CRISPR-Cas , Carica , Técnicas de Amplificación de Ácido Nucleico , Plantas Modificadas Genéticamente , Carica/genética , Carica/virología , Sistemas CRISPR-Cas/genética , Plantas Modificadas Genéticamente/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Potyvirus/genética , Potyvirus/aislamiento & purificación , Recombinasas/metabolismo , Límite de Detección , Proteínas Bacterianas , Endodesoxirribonucleasas , Proteínas Asociadas a CRISPR

RESUMEN

White yam (Dioscorea rotundata) plants collected from farmers' fields and planted at the Areka Agricultural Research Center, Southern Ethiopia, displayed mosaic, mottling, and chlorosis symptoms. To determine the presence of viral pathogens, an investigation for virome characterization was conducted by Illumina high-throughput sequencing. The bioinformatics analysis allowed the assembly of five viral genomes, which according to the ICTV criteria were assigned to a novel potyvirus (3 genome sequences) and a novel crinivirus (2 genome sequences). The potyvirus showed ~ 66% nucleotide (nt) identity in the polyprotein sequence to yam mosaic virus (NC004752), clearly below the demarcation criteria of 76% identity. For the crinivirus, the RNA 1 and RNA 2 shared the highest sequence identity to lettuce chlorosis virus, and alignment of the aa sequence of the RdRp, CP and HSP70h (~ 49%, 45% and 76% identity), considered for the demarcation criteria, revealed the finding of a novel virus species. The names Ethiopian yam virus (EYV) and Yam virus 1 (YV-1) are proposed for the two tentative new virus species.

Asunto(s)

Crinivirus , Dioscorea , Genoma Viral , Filogenia , Enfermedades de las Plantas , Potyvirus , Dioscorea/virología , Potyvirus/genética , Potyvirus/aislamiento & purificación , Potyvirus/clasificación , Etiopía , Enfermedades de las Plantas/virología , Crinivirus/genética , Crinivirus/aislamiento & purificación , Crinivirus/clasificación , Genoma Viral/genética , ARN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Coinfección/virología

RESUMEN

Papaya ringspot virus (PRSV) limits papaya production worldwide. Previously, we generated transgenic lines of hybrid Tainung No.2 (TN-2) carrying the coat protein (CP) gene of PRSV with broad resistance to PRSV strains. Unfortunately, all of them were female, unacceptable for growers and consumers in practical applications. With our reported flanking sequences and the newly released papaya genomic information, the CP-transgene insert was identified at a non-coding region in chromosome 3 of the papaya genome, and the flanking sequences were verified and extended. The female transgenic line 16-0-1 was first used for backcrossing with the parental Sunrise cultivar six times and then followed by selfing three times. With multi-level molecular markers developed from the PRSV CP transgene and the genomic flanking sequences, the presence and zygosity of the CP transgene were characterized at the seedling stage. Meanwhile, hermaphrodite genotype was identified by a sex-linked marker. With homozygotic transgene and horticultural properties of Sunrise, a selected hermaphrodite individual was propagated by tissue culture (TC) and used as maternal progenitor to cross with non-transgenic parental cultivar Thailand to generate a new hybrid cultivar TN-2 with a hemizygotic CP-transgene. Three selected hermaphrodite individuals of transgenic TN were micropropagated by TC, and they showed broad-spectrum resistance to different PRSV strains from Taiwan, Hawaii, Thailand, and Mexico under greenhouse conditions. The selected clone TN-2 #1, with excellent horticultural traits, also showed complete resistance to PRSV under field conditions. These selected TC clones of hermaphrodite transgenic TN-2 provide a novel cultivation system in Taiwan and elsewhere.

Asunto(s)

Proteínas de la Cápside , Carica , Resistencia a la Enfermedad , Enfermedades de las Plantas , Plantas Modificadas Genéticamente , Potyvirus , Transgenes , Carica/virología , Carica/genética , Potyvirus/genética , Plantas Modificadas Genéticamente/virología , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/virología , Proteínas de la Cápside/genética , Genoma de Planta , Mapeo Cromosómico

RESUMEN

The RNA regulatory network is a complex and dynamic regulation in plant cells involved in mRNA modification, translation, and degradation. Ras-GAP SH3 domain-binding protein (G3BP) is a scaffold protein for the assembly of stress granules (SGs) and is considered an antiviral component in mammals. However, the function of G3BP during virus infection in plants is still largely unknown. In this study, four members of the G3BP-like proteins (NtG3BPLs) were identified in Nicotiana tabacum and the expression levels of NtG3BPL1 were upregulated during chilli veinal mottle virus (ChiVMV) infection. NtG3BPL1 was localized in the nucleus and cytoplasm, forming cytoplasmic granules under transient high-temperature treatment, whereas the abundance of cytoplasmic granules was decreased under ChiVMV infection. Overexpression of NtG3BPL1 inhibited ChiVMV infection and delayed the onset of symptoms, whereas knockout of NtG3BPL1 promoted ChiVMV infection. In addition, NtG3BPL1 directly interacted with ChiVMV 6K2 protein, whereas 6K2 protein had no effect on NtG3BPL1-derived cytoplasmic granules. Further studies revealed that the expression of NtG3BPL1 reduced the chloroplast localization of 6K2-GFP and the NtG3BPL1-6K2 interaction complex was localized in the cytoplasm. Furthermore, NtG3BPL1 promoted the degradation of 6K2 through autophagy pathway, and the accumulation of 6K2 and ChiVMV was affected by autophagy activation or inhibition in plants. Taken together, our results demonstrate that NtG3BPL1 plays a positive role in tobacco resistance against ChiVMV infection, revealing a novel mechanism of plant G3BP in antiviral strategy.

Asunto(s)

Nicotiana , Enfermedades de las Plantas , Proteínas de Plantas , Nicotiana/virología , Nicotiana/genética , Nicotiana/metabolismo , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente , Potyvirus/fisiología