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Tomato leaf curl New Delhi virus (ToLCNDV) is a begomovirus causing significant melon (Cucumis melo) crop losses globally. This study aims to map the ToLCNDV resistance in the PI 414723 melon accession, previously identified and characterized through phenotypic studies, thereby exploring shared genomic regions with the established resistant source WM-7. In the present study, WM-7 and PI 414723 were crossed with the susceptible accessions 'Rochet' and 'Blanco' respectively, to generate F1 hybrids. These hybrids were self-pollinated to generate the populations for mapping the ToLCNDV resistance region and designing markers for marker-assisted selection. Disease evaluation included visual symptom scoring, viral-load quantification and tissue printing. Genotyping-by-sequencing and SNP markers were used for quantitative trait loci (QTL) mapping. For genetic analysis, qPCR and bulked segregant RNA-seq (BSR-seq) were performed. Gene expression was assessed using RNA-seq, and qRT-PCR was used for confirmation. The research narrows the candidate region for resistance in WM-7 and identifies overlapping QTLs on chromosome 11 in PI 414723, found in the region of the DNA primase large subunit. BSR-seq and expression analyses highlight potential regulatory roles of chromosome 2 in conferring resistance. Differential expression was confirmed for six genes in the candidate region on chromosome 2. This study confirms the existence of common resistance genes in PI 414723 and WM-7.

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In early spring 2018, significant mosaic disease symptoms were observed for the first time on barley leaves (Hordeum vulgare L., cv. New Sachiho Golden) in Takanezawa, Tochigi Prefecture, Japan. This cultivar carries the resistance gene rym3 (rym; resistance to yellow mosaic). Through RNA-seq analysis, Barley yellow mosaic virus (BaYMV-Takanezawa) was identified in the roots of all five plants (T01-T05) in the field. Phylogenetic analysis of RNA1, encompassing known BaYMV pathotypes I through V, revealed that it shares the same origin as isolate pathotype IV (BaYMV-Ohtawara pathotype). However, RNA2 analysis of isolates revealed the simultaneous presence of two distinct BaYMV isolates, BaYMV-Takanezawa-T01 (DRR552862, closely related to pathotype IV) and BaYMV-Takanezawa-T02 (DRR552863, closely related to pathotype III). The amino acid sequences of the BaYMV-Takanezawa isolates displayed variations, particularly in the VPg and N-terminal region of CP, containing mutations not found in other domains of the virus genome. Changes in the CI (RNA1 amino acid residue 459) and CP (RNA1 amino acid residue 2138) proteins correlated with pathogenicity. These findings underscore the importance of monitoring and understanding the genetic diversity of BaYMV for effective disease management strategies in crop breeding.

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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.

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Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

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Viral diseases are crucial determinants affecting tobacco cultivation, leading to a substantial annual decrease in production. Previous studies have demonstrated the regulatory function of the C3HC4 family of plant zinc finger proteins in combating bacterial diseases. However, it remains to be clarified whether this protein family also plays a role in regulating resistance against plant viruses. In this study, the successful cloning of the zinc finger protein coding gene NbZFP1 from Nicotiana benthamiana has been achieved. The full-length coding sequence of NbZFP1 is 576 bp. Further examination and analysis of this gene revealed its functional properties. The induction of NbZFP1 transcription in N. benthamiana has been observed in response to TMV, CMV, and PVY. Transgenic N. benthamiana plants over-expressing NbZFP1 demonstrated a notable augmentation in the production of chlorophyll a (P < 0.05). Moreover, NbZFP1-overexpressing tobacco exhibited significant resistance to TMV, CMV, and PVY, as evidenced by a decrease in virus copies (P < 0.05). In addition, the defense enzymes activities of PAL, POD, and CAT experienced a significant increase (P < 0.05). The up-regulated expression of genes of NbPAL, NbNPR1 and NbPR-1a, which play a crucial role in SA mediated defense, indicated that the NbZFP1 holds promise in enhancing the virus resistance of tobacco plant. Importantly, the results demonstrate that NbZFP1 can be considered as a viable candidate gene for the cultivation of crops with enhanced virus resistance.

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Introduction: Grapevine (Vitis vinifera) is an important fruit crop which contributes significantly to the agricultural sector worldwide. Grapevine viruses are widespread and cause serious diseases which impact the quality and quantity of crop yields. More than 80 viruses plague grapevine, with RNA viruses constituting the largest of these. A recent extension to the clustered regularly interspaced, short palindromic repeat (CRISPR) armory is the Cas13 effector, which exclusively targets single-strand RNA. CRISPR/Cas has been implemented as a defense mechanism in plants, against both DNA and RNA viruses, by being programmed to directly target and cleave the viral genomes. The efficacy of the CRISPR/Cas tool in plants is dependent on efficient delivery of its components into plant cells. Methods: To this end, the aim of this study was to use the recent Cas13d variant from Ruminococcus flavefaciens (CasRx) to target the RNA virus, grapevine virus A (GVA). GVA naturally infects grapevine, but can infect the model plant Nicotiana benthamiana, making it a helpful model to study virus infection in grapevine. gRNAs were designed against the coat protein (CP) gene of GVA. N. benthamiana plants expressing CasRx were co-infiltrated with GVA, and with a tobacco rattle virus (TRV)-gRNA expression vector, harbouring a CP gRNA. Results and discussion: Results indicated more consistent GVA reductions, specifically gRNA CP-T2, which demonstrated a significant negative correlation with GVA accumulation, as well as multiple gRNA co-infiltrations which similarly showed reduced GVA titre. By establishing a virus-targeting defense system in plants, efficient virus interference mechanisms can be established and applied to major crops, such as grapevine.

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The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

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Microbiota are known to modulate the host response to influenza infection, but the mechanisms remain largely unknown. Gut metabolites are the key mediators through which gut microbes play anti-influenza effect. Transferring fecal metabolites from mice with high influenza resistance into antibiotic-treated recipient mice conferred resistance to influenza infections. By comparing the metabolites of different individuals with high or low influenza resistance, we identified and validated N-acetyl-D-glucosamine (GlcNAc) and adenosine showed strong positive correlations with influenza resistance and exerted anti-influenza effects in vivo or in vitro, respectively. Especially, GlcNAc mediated the anti-influenza effect by increasing the proportion and activity of NK cells. Several gut microbes, including Clostridium sp., Phocaeicola sartorii, and Akkermansia muciniphila, were positively correlated with influenza resistance, and can upregulate the level of GlcNAc in the mouse gut by exogenous supplementation. Subsequent studies confirmed that administering a combination of the three bacteria to mice via gavage resulted in similar modulation of NK cell responses as observed with GlcNAc. This study demonstrates that gut microbe-produced GlcNAc protects the host against influenza by regulating NK cells, facilitating the elucidation of the action mechanism of gut microbes mediating host influenza resistance.

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Plant viruses are the main pathogens which cause significant quality and yield losses in tomato crops. The important viruses that infect tomatoes worldwide belong to five genera: Begomovirus, Orthotospovirus, Tobamovirus, Potyvirus, and Crinivirus. Tomato resistance genes against viruses, including Ty gene resistance against begomoviruses, Sw gene resistance against orthotospoviruses, Tm gene resistance against tobamoviruses, and Pot 1 gene resistance against potyviruses, have been identified from wild germplasm and introduced into cultivated cultivars via hybrid breeding. However, these resistance genes mainly exhibit qualitative resistance mediated by single genes, which cannot protect against virus mutations, recombination, mixed-infection, or emerging viruses, thus posing a great challenge to tomato antiviral breeding. Based on the epidemic characteristics of tomato viruses, we propose that future studies on tomato virus resistance breeding should focus on rapidly, safely, and efficiently creating broad-spectrum germplasm materials resistant to multiple viruses. Accordingly, we summarized and analyzed the advantages and characteristics of the three tomato antiviral breeding strategies, including marker-assisted selection (MAS)-based hybrid breeding, RNA interference (RNAi)-based transgenic breeding, and CRISPR/Cas-based gene editing. Finally, we highlighted the challenges and provided suggestions for improving tomato antiviral breeding in the future using the three breeding strategies.

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Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.

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Plumeria pudica, known as bridal bouquet, exhibiting characteristic symptoms of orthotospovirus infection were found in different localities in Brazil. Symptoms were restricted to leaves of the middle and lower thirds of a few branches of each plant. Electron microscopy, molecular analyses, and complete genome sequencing identified the orthotospovirus as groundnut ringspot virus (GRSV),member of the species Orthotospovirus arachianuli. The virus was poorly transmitted mechanically to P. pudica. Reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses performed using total RNA extracted from leaf blades, primary veins, petioles, and regions of petiole insertion on branches indicated the presence of GRSV, predominantly in the symptomatic leaf blades. Symptomatic branches propagate vegetatively, often resulting in plants expressing GRSV symptoms. In contrast, vegetative propagation of the asymptomatic branches of infected plants predominantly generates plants without GRSV symptoms. The resistance of P. pudica plants to GRSV infection, restricted systemic viral movement, and expression of symptoms in infected plants suggest that this orthotospovirus does not threaten this ornamental plant.

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Tomato leaf curl Bangalore virus (ToLCBaV) is one of the most important plant viruses. The infection causes substantial yield losses in tomato crop. The current viral disease management is based mainly on introgression of Ty locus into new tomato cultivars. Unfortunately, strains of the leaf curl virus have been evolving and are breaking Ty based tolerance in tomato. In this study, the defence response to ToLCBaV infection has been compared between contrasting tomato genotypes, resistant line (IIHR 2611; without any known Ty markers) and the susceptible line (IIHR 2843). We carried out comparative transcriptome profiling, and gene expression analysis in an effort to identify gene networks that are associated with a novel ToLCBaV resistance. A total of 22,320 genes were examined to identify differentially expressed genes (DEGs). We found that 329 genes of them were expressed significantly and differentially between ToLBaV-infected samples of both IIHR 2611 and IIHR 2843. A good number of DEGs were related to defence response, photosynthesis, response to wounding, toxin catabolic process, glutathione metabolic process, regulation of transcription DNA-template, transcription factor activity, and sequence-specific DNA binding. A few selected genes such as, nudix hydrolase 8, MIK 2-like, RING-H2 finger protein ATL2-like, MAPKKK 18-like, EDR-2, SAG 21 wound-induced basic protein, GRXC6 and P4 were validated using qPCR. The pattern of gene expression was significantly different in resistant and susceptible plants during disease progression. Both positive and negative regulators of virus resistance were found in the present study. These findings will facilitate breeding and genetic engineering efforts to incorporate novel sources of ToLCBaV resistance in tomatoes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03629-5.

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Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

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Control of plant virus diseases is a big challenge in agriculture as is resistance in plant lines to infection by viruses. Recent progress using advanced technologies has provided fast and durable alternatives. One of the most promising techniques against plant viruses that is cost-effective and environmentally safe is RNA silencing or RNA interference (RNAi), a technology that could be used alone or along with other control methods. To achieve the goals of fast and durable resistance, the expressed and target RNAs have been examined in many studies, with regard to the variability in silencing efficiency, which is regulated by various factors such as target sequences, target accessibility, RNA secondary structures, sequence variation in matching positions, and other intrinsic characteristics of various small RNAs. Developing a comprehensive and applicable toolbox for the prediction and construction of RNAi helps researchers to achieve the acceptable performance level of silencing elements. Although the attainment of complete prediction of RNAi robustness is not possible, as it also depends on the cellular genetic background and the nature of the target sequences, some important critical points have been discerned. Thus, the efficiency and robustness of RNA silencing against viruses can be improved by considering the various parameters of the target sequence and the construct design. In this review, we provide a comprehensive treatise regarding past, present and future prospective developments toward designing and applying RNAi constructs for resistance to plant viruses.

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Environmental changes and global warming may promote the emergence of unknown viruses, whose spread is favored by the trade in plant products. Viruses represent a major threat to viticulture and the wine industry. Their management is challenging and mostly relies on prophylactic measures that are intended to prevent the introduction of viruses into vineyards. Besides the use of virus-free planting material, the employment of agrochemicals is a major strategy to prevent the spread of insect vectors in vineyards. According to the goal of the European Green Deal, a 50% decrease in the use of agrochemicals is expected before 2030. Thus, the development of alternative strategies that allow the sustainable control of viral diseases in vineyards is strongly needed. Here, we present a set of innovative biotechnological tools that have been developed to induce virus resistance in plants. From transgenesis to the still-debated genome editing technologies and RNAi-based strategies, this review discusses numerous illustrative studies that highlight the effectiveness of these promising tools for the management of viral infections in grapevine. Finally, the development of viral vectors from grapevine viruses is described, revealing their positive and unconventional roles, from targets to tools, in emerging biotechnologies.

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Rose rosette disease (RRD), caused by the rose rosette emaravirus (RRV), is a major viral disease in roses (Rosa sp.) that threatens the rose industry. Recent studies have revealed quantitative trait loci (QTL) for reduced susceptibility to RRD in the linkage groups (LGs) 1, 5, 6, and 7 in tetraploid populations and the LGs 1, 3, 5, and 6 in diploid populations. In this study, we seek to better localize and understand the relationship between QTL identified in both diploid and tetraploid populations. We do so by remapping the populations found in these studies and performing a meta-analysis. This analysis reveals that the peaks and intervals for QTL using diploid and tetraploid populations co-localized on LG 1, suggesting that these are the same QTL. The same was seen on LG 3. Three meta-QTL were identified on LG 5, and two were discovered on LG 6. The meta-QTL on LG 1, MetaRRD1.1, had a confidence interval (CI) of 10.53 cM. On LG 3, MetaRRD3.1 had a CI of 5.94 cM. MetaRRD5.1 had a CI of 17.37 cM, MetaRRD5.2 had a CI of 4.33 cM, and MetaRRD5.3 had a CI of 21.95 cM. For LG 6, MetaRRD6.1 and MetaRRD6.2 had CIs of 9.81 and 8.81 cM, respectively. The analysis also led to the identification of potential disease resistance genes, with a primary interest in genes localized in meta-QTL intervals on LG 5 as this LG was found to explain the greatest proportion of phenotypic variance for RRD resistance. The results from this study may be used in the design of more robust marker-based selection tools to track and use a given QTL in a plant breeding context.

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CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.

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Aging of the immune system involves functional changes in individual cell populations, in hematopoietic tissues and at the systemic level. They are mediated by factors produced by circulating cells, niche cells, and at the systemic level. Age-related alterations in the microenvironment of the bone marrow and thymus cause a decrease in the production of naive immune cells and functional immunodeficiencies. Another result of aging and reduced tissue immune surveillance is the accumulation of senescent cells. Some viral infections deplete adaptive immune cells, increasing the risk of autoimmune and immunodeficiency conditions, leading to a general degradation in the specificity and effectiveness of the immune system in old age. During the COVID-19 pandemic, the state-of-the-art application of mass spectrometry, multichannel flow cytometry, and single-cell genetic analysis have provided vast data on the mechanisms of aging of the immune system. These data require systematic analysis and functional verification. In addition, the prediction of age-related complications is a priority task of modern medicine in the context of the increase in the aged population and the risk of premature death during epidemics. In this review, based on the latest data, we discuss the mechanisms of immune aging and highlight some cellular markers as indicators of age-related immune disbalance that increase the risk of senile diseases and infectious complications.

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Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

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Vacuolar ATPases (V-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; OsV-ATPase subunit d (OsV-ATPase d) is part of an integral, membrane-embedded V0 complex in the V-ATPase complex. Whether OsV-ATPase d is involved in phytohormone biosynthesis and resistance in rice remains unknown. The knockout mutants of OsV-ATPase d in rice were generated using the CRISPR/Cas9 system, and mutation of OsV-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed that OsV-ATPase d is probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Compared to wild type, mutation of OsV-ATPase d significantly increased JA and ABA biosynthesis and resistance against Southern rice black-streaked dwarf virus (SRBSDV), but it decreased resistance against Rice stripe virus (RSV) in rice. The data presented in this study reveal that OsV-ATPase d mediates phytohormone biosynthesis and virus resistance in rice and can be selected as a potential target for resistance breeding in rice.