Integrative analysis of the transcriptome and metabolome reveals the response mechanism to tomato spotted wilt virus

Tomato spotted wilt virus(TSWV)is an important virus that has rapidly spread throughout the world.TSWV seriously hinders the production of tomato(Solanum lycopersicum)and other plants.In order to discover more new genes and metabolites related to TSWV resistance in tomato plants,the genes and metabolites related to the resistance of tomato plants inoculated with TSWV were identified and studied herein.The tomato TSWV-resistance line YNAU335(335)and TSWV-susceptible lines NO5 and 96172I(961)were used as the transcriptome and metabolome research materials.Transcriptomic and metabolomic techniques were used to analyze the gene and metabolite response mechanisms to TSWV inoculation.A total of 3566,2951,and 2674 differentially expressed genes(DEGs)were identified in lines 335,NO5,and961,respectively.Meanwhile,208,228,and 273 differentially accumulated metabolites(DAMs)were identified in lines 335,NO5,and 961,respectively.In line 335,the number of DEGs was the highest,but the number of DAMs was lowest.Furthermore,903 DEGs and 94 DAMs were common to the response to TSWV in the three inbred lines.The 903 DEGs and 94 DAMs were mainly enriched in the plant hormone signal transduction and flavonoid synthesis pathways.In addition,many nucleotide-binding site-leucine-rich repeat genes and transcription factors were found that might be involved in the TSWV response.These results provide new insights into TSWV resistance mechanisms.

Population structure and association mapping to detect QTL controlling tomato spotted wilt virus resistance in cultivated peanuts

Tomato spotted wilt(TSW)is a serious virus disease of peanut in the United States.Breeding for TSWV resistance would be facilitated by the implementation of marker-assisted selection in breeding programs;however,genes associated with resistance have not been identified.Association mapping is a type of genetic mapping that can exploit relationships between markers and traits in many lineages.The objectives of this study were to examine genetic diversity and population structure in the U.S.peanut mini-core collection using simple sequence repeat(SSR)markers,and to conduct association mapping between SSR markers and TSWV resistance in cultivated peanuts.One hundred and thirty-three SSR markers were used for genotyping 104 accessions.Four subpopulations,generally corresponding to botanical varieties,were classified by population structure analysis.Association mapping analysis indicated that five markers:pP GPseq5D5,GM1135,GM1991,TC23C08,and TC24C06,were consistently associated with TSW resistance by the Q,PCA,Q+K,and PCA+K models.These markers together explained 36.4%of the phenotypic variance.Moreover,pP GPseq5D5 and GM1991 were associated with both visual symptoms of TSWV and ELISA values with a high R^2.The potential of these markers for use in a marker-assisted selection program to breed peanut for resistance to TSWV is discussed.

Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable mutations.Because many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

Identification of ExpIdentification of Expressed Resistance Gene Analogs from Peanut (Arachis hypogaea L.) Expressed Sequence Tags

Low genetic diversity makes peanut （Arachis hypogaea L.） very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat （NBS-LRR） resistance genes （R） have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs （RGAs） from peanut expressed sequence tags （ESTs） and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats （SSRs） were identified from 25 peanut expressed RGAs. One SSR polymorphic marker （RGA121） was identified. Two polymerase chain reaction-based markers （Ahsw-1 and Ahsw-2） developed from RGA013 were homologous to the Tomato Spotted Wilt Virus （TSWV） resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.