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Key messageA begomovirus resistance gene pepy-1, which encodes the messenger RNA surveillance factor Pelota, was identified in pepper (C. annuum) through map-based cloning and functional characterization.Pepper yellow leaf curl disease caused by begomoviruses seriously affects pepper (Capsicum spp.) production in a number of regions around the world. Ty genes of tomato, which confer resistance to the tomato yellow leaf curl virus, are the only begomovirus resistance genes cloned to date. In this study, we focused on the identification of begomovirus resistance genes in Capsicum annuum. BaPep-5 was identified as a novel source of resistance against pepper yellow leaf curl Indonesia virus (PepYLCIV) and pepper yellow leaf curl Aceh virus (PepYLCAV). A single recessive locus, which we named as pepper yellow leaf curl disease virus resistance 1 (pepy-1), responsible for PepYLCAV resistance in BaPep-5 was identified on chromosome 5 in an F2 population derived from a cross between BaPep-5 and the begomovirus susceptible accession BaPep-4. In the target region spanning 34 kb, a single candidate gene, the messenger RNA surveillance factor Pelota, was identified. Whole-genome resequencing of BaPep-4 and BaPep-5 and comparison of their genomic DNA sequences revealed a single nucleotide polymorphism (A to G) located at the splice site of the 9th intron of CaPelota in BaPep-5, which caused the insertion of the 9th intron into the transcript, resulting in the addition of 28 amino acids to CaPelota protein without causing a frameshift. Virus-induced gene silencing of CaPelota in the begomovirus susceptible pepper No.218 resulted in the gain of resistance against PepYLCIV, a phenotype consistent with BaPep-5. The DNA marker developed in this study will greatly facilitate marker-assisted breeding of begomovirus resistance in peppers.

Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

Yellow leaf curl disease caused by begomoviruses has emerged as a major limitation to the production of eggplant (Solanum melongena) in several regions of the world. Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) is a bipartite begomovirus isolated from diseased eggplant in the areas of the Indochinese peninsula, South China, and Maritime Southeast Asia. In this study, five begomovirus species isolated in Indonesia, including TYLCKaV, were inoculated to eggplant using infectious clones. Among the inoculated begomoviruses, TYLCKaV alone induced typical yellowing and curling symptoms in eggplant. Inoculation of TYLCKaV to a panel of crop species (eggplant, tomato, pepper, tobacco, cucumber, melon, and squash) commonly grown in Indonesia clarified that TYLCKaV infects and causes yellow leaf curl disease mainly in eggplant and tomato. Agroinfiltration of TYLCKaV to 736 accessions of eggplant enabled selection of 33 resistant candidates. Further evaluation by TYLCKaV graft-inoculation of the resistant candidates clarified that 10 accessions were classifiable as moderately resistant and 23 accessions as highly resistant. Among the highly resistant accessions, 22 accessions restricted viral DNA accumulation to a significantly lower amount than the susceptible control. These 22 highly resistant accessions represent valuable genetic resources for breeding begomovirus resistance in eggplant.

Three primary species from the Begomovirus genus, Pepper yellow leaf curl Indonesia virus (PepYLCIV), Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), and Tomato leaf curl New Delhi virus (ToLCNDV), are suspected of spreading throughout pepper production centers, and plants are infected by a single species or a combination of two or three species. This study was conducted to provide complete information about the symptoms, incidence and severity, whitefly biotypes, as well as the dominance status of the three Begomovirus species in pepper-producing areas in Java. A DNA analysis was carried out on leaf samples to identify Begomovirus species and biotypes of B. tabaci collected from 18 areas (16 districts) in lowlands (<400 m asl) and highlands (>700 m asl). The DNA analysis showed that B. tabaci biotype B was the most commonly detected in all locations compared to the A, AN, and Q biotypes. The incidence of begomovirus infection was at a high level, 93% and 88.78% in the lowlands and highlands, respectively. However, the severity of begomovirus infection was significantly higher in the lowlands (54.50%) than in the highlands (38.11%). A single infection of PepYLCIV was most dominant in all locations sampled and caused severe infection, followed by a mixed infection with TYLCKaV. Therefore, the current status of begomovirus infection, especially PepYLCIV, can provide advice to farmers using more tolerant and resistant varieties as well as a breeding strategy for resistant pepper varieties.

One important factor for successful disease management is the ability to rapidly and accurately identify the causal agent. Plant viruses cause severe economic losses and pose a serious threat to sustainable agriculture. Therefore, optimization of the speed, sensitivity, feasibility, portability, and accuracy of virus detection is urgently needed. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid diagnostic method utilizing the CRISPR–Cas12a system for detecting two geminiviruses, tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV), which have single-stranded DNA genomes. Our assay detected TYLCV and ToLCNDV in infected plants with high sensitivity and specificity. Our newly developed assay can be performed in ~1 h and provides easy-to-interpret visual readouts using a simple, low-cost fluorescence visualizer, making it suitable for point-of-use applications.

Pepper yellow leaf curl disease caused by begomoviruses has emerged as a major limitation to the cultivation of pepper (Capsicum spp.) production in a number of regions around the world. Although begomovirus resistance has been reported in several accessions, pepy-1 derived from BaPep-5 (C. annuum) is the only begomovirus resistance gene cloned to date. In this study, we evaluated the resistance of a C. chinense accession GR1 against pepper yellow leaf curl Indonesia virus (PepYLCIV), which is one of the predominant begomovirus species infecting pepper plants in Indonesia. Infection of PepYLCIV to susceptible Habanero (C. chinense) plants induced typical yellowing symptoms, whereas the GR1 plant showed symptoms with very low severity. Moreover, the accumulation of viral DNA was restricted in GR1 compared to Habanero. Phenotypic analyses of F1 and F2 populations obtained by crossing Habanero with GR1 inferred that the resistance is a dominant trait controlled by multiple genes. Linkage analysis in the F2 population using restriction site-associated DNA sequencing data detected two significant quantitative trait loci (QTLs), one on chromosome 4 and another on chromosome 11, which explained 31.6 and 19.7% of the phenotypic variation, respectively. Moreover, QTL-seq conducted using F3 population partially confirmed the result of F2 population, which detected QTLs on chromosome 3 and 11, respectively. The QTLs identified for PepYLCIV resistance in this research are novel since no other resistance causing QTLs have ever been reported in these genomic regions. GR1 is a highly valuable genetic resource for the breeding of begomovirus resistance in peppers.

The selection of resistant crops is an effective method for controlling geminivirus diseases. ty-5 encodes a messenger RNA surveillance factor Pelota with a single amino acid mutation (PelotaV16G), which confers effective resistance to tomato yellow leaf curl virus (TYLCV). No studies have investigated whether ty-5 confers resistance to other geminiviruses. Here, we demonstrate that the tomato ty-5 line exhibits effective resistance to various geminiviruses. It confers resistance to two representative begomoviruses, tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite complex and tomato leaf curl Yunnan virus. The ty-5 line also exhibits partial resistance to a curtovirus beet curly top virus. Importantly, ty-5 confers resistance to TYLCV with a betasatellite. Southern blotting and quantitative polymerase chain reaction analyses showed that significantly less DNA of these geminiviruses accumulated in the ty-5 line than in the susceptible line. Moreover, knockdown of Pelota expression converted a Nicotiana benthamiana plant from a geminivirus-susceptible host to a geminivirus-resistant host. Overall, our findings suggest that ty-5 is an important resistance gene resource for crop breeding to control geminiviruses.

With climate warming, drought becomes a vital challenge for agriculture. Extended drought periods affect plant–pathogen interactions. We demonstrate an interplay in tomato between drought and infection with tomato yellow leaf curl virus (TYLCV). Infected plants became more tolerant to drought, showing plant readiness to water scarcity by reducing metabolic activity in leaves and increasing it in roots. Reallocation of osmolytes, such as carbohydrates and amino acids, from shoots to roots suggested a role of roots in protecting infected tomatoes against drought. To avoid an acute response possibly lethal for the host organism, TYLCV down‐regulated the drought‐induced activation of stress response proteins and metabolites. Simultaneously, TYLCV promoted the stabilization of osmoprotectants' patterns and water balance parameters, resulting in the development of buffering conditions in infected plants subjected to prolonged stress. Drought‐dependent decline of TYLCV amounts was correlated with HSFA1‐controlled activation of autophagy, mostly in the roots. The tomato response to combined drought and TYLCV infection points to a mutual interaction between the plant host and its viral pathogen. Tomato yellow leaf curl virus infection is able to extend the survival of tomato plants subjected to drought, mainly by promoting the stabilization of stress markers and by reallocating important osmoprotective metabolites from shoots to roots.

Key messageTy-6 is a major resistance gene on chromosome 10 of tomato that provides resistance against monopartite and bipartite begomoviruses and complements resistance conferred by the known Ty-3 and ty-5 genes.Resistance to monopartite and bipartite begomoviruses is an important breeding objective for cultivated tomato. Several begomovirus resistance genes have been introgressed from related Solanum species and are available for breeding purposes. In the present study, we mapped an additional locus, Ty-6, to chromosome 10 of tomato. Ty-6 is effective against both monopartite Tomato yellow leaf curl virus (TYLCV) and bipartite Tomato mottle virus (ToMoV). Gene action is incomplete dominance, with an intermediate resistance response when Ty-6 is heterozygous. Analysis of populations segregating for Ty-6 along with Ty-3 or ty-5 indicates that the highest level of resistance against TYLCV is attained when Ty-6 is combined with an additional resistance allele. Our results also demonstrate that ty-5 is ineffective against ToMoV. Although multiple SNPs linked to Ty-6 were identified and can be used for breeding purposes, none of these were consistently polymorphic between Ty-6 and ty-6 breeding lines. Further research is underway to generate resequencing data for several Ty-6 inbred lines for the discovery of additional sequence polymorphisms that can be used for fine mapping and characterizing the Ty-6 locus.

Actinomycetes comprise a large group of biocontrol bacteria, yet no studies have reported on their effects against plant virus diseases. Here, we evaluated the control effects and the possible mechanisms of Streptomyces pactum Act12 against tomato yellow leaf curl virus disease (TYLCD) through field and pot experiments. We also analyzed changes in plant growth and rhizosphere microbial community composition following the application of Act12. The pre-inoculation of soil with the Act12 agent enhanced the activities of peroxidase and chitinase while upregulating the expression of genes related to plant systemic resistance (PR-1 and SIPI-II) and specific TYLCD resistance (SIPer1 and SIVRSLip) in tomato leaves under field conditions. The effects of Act12 at inducing a decline in TYLCD severity and promoting plant growth were deemed satisfactory in all three field experiments. In the pot experiments, Act12 induced a decline in the viral DNA level and an enhancing of peroxidase, polyphenoloxidase, and phenylalanine ammonia lyase activities in tomato leaves. Additionally, Act12 treatment reduced the accumulation of H2O2 and O·−2, increased the levels of salicylic and jasmonic acids, and upregulated the expression of genes related to plant resistance and RNA interference in tomato leaves. Applying Act12 also increased the diversity of bacteria and the abundance of potential beneficial microbes (e.g., Trichoderma and Bacillus) in the root-zone and root-surface soils, resulting in modular differentiation of co-occurrence networks. In conclusion, applying the S. pactum Act12 agent reduced TYLCD severity and promoted plant growth in tomato. Whether the changes of resistance-related gene expression and rhizosphere microbial community composition contribute to TYLCV resistance needs to be further investigated.