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Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter.

ABSTRACT: TBR225 is one of the most popular commercial rice varieties in Northern Vietnam. However, this variety is highly susceptible to bacterial leaf blight (BLB), a disease caused by Xanthomonas oryzae pv. oryzae (Xoo) which can lead to important yield losses. OsSWEET14 belongs to the SWEET gene family that encodes sugar transporters. Together with other Clade III members, it behaves as a susceptibility (S) gene whose induction by Asian Xoo Transcription-Activator-Like Effectors (TALEs) is absolutely necessary for disease. In this study, we sought to introduce BLB resistance in the TBR225 elite variety. First, two Vietnamese Xoo strains were shown to up-regulate OsSWEET14 upon TBR225 infection. To investigate if this induction is connected with disease susceptibility, nine TBR225 mutant lines with mutations in the AvrXa7, PthXo3 or TalF TALEs DNA target sequences of the OsSWEET14 promoter were obtained using the CRISPR/Cas9 editing system. Genotyping analysis of T0 and T1 individuals showed that mutations were stably inherited. None of the examined agronomic traits of three transgene-free T2 edited lines were significantly different from those of wild-type TBR225. Importantly, one of these T2 lines, harboring the largest homozygous 6-bp deletion, displayed decreased OsSWEET14 expression as well as a significantly reduced susceptibility to a Vietnamese Xoo strains and complete resistance to another one. Our findings indicate that CRISPR/Cas9 editing conferred an improved BLB resistance to a Vietnamese commercial elite rice variety.

SUBMITTER: Duy PN

PROVIDER: S-EPMC8428762 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Major biotic stresses viz., bacterial blight (BB) and blast and brown plant hopper (BPH) coupled with abiotic stresses like drought stress, significantly affect rice yields. To address this, marker-assisted intercross (IC) breeding involving multiple donors was used to combine three BB resistance genes-xa5, xa13 and Xa21, two blast resistance genes-Pi9 and Pi54, two BPH resistance genes-Bph20 and Bph21, and four drought tolerant quantitative trait loci (QTL)-qDTY1.1, qDTY2.1, qDTY3.1 and qDTY12.1-in the genetic background of the elite Indian rice cultivar 'Krishna Hamsa'. Three cycles of selective intercrossing followed by selfing coupled with foreground selection and phenotyping for the target traits resulted in the development of 196 introgression lines (ILs) with a myriad of gene/QTL combinations. Based on the phenotypic reaction, the ILs were classified into seven phenotypic classes of resistance/tolerance to the following: (1) BB, blast and drought-5 ILs; (2) BB and blast-10 ILs; (3) BB and drought-9 ILs; (4) blast and drought-42 ILs; (5) BB-3 ILs; (6) blast-84 ILs; and (7) drought-43 ILs; none of the ILs were resistant to BPH. Positive phenotypic response (resistance) was observed to both BB and blast in 2 ILs, BB in 9 ILs and blast in 64 ILs despite the absence of corresponding R genes. Inheritance of resistance to BB and/or blast in such ILs could be due to the unknown genes from other parents used in the breeding scheme. Negative phenotypic response (susceptibility) was observed in 67 ILs possessing BB-R genes, 9 ILs with blast-R genes and 9 ILs harboring QTLs for drought tolerance. Complex genic interactions and recombination events due to the involvement of multiple donors explain susceptibility in some of the marker positive ILs. The present investigation successfully demonstrates the possibility of rapid development of multiple stress-tolerant/resistant ILs in the elite cultivar background involving multiple donors through selective intercrossing and stringent phenotyping. The 196 ILs in seven phenotypic classes with myriad of gene/QTL combinations will serve as a useful genetic resource in combining multiple biotic and abiotic stress resistance in future breeding programs.

Project description:Fusarium head blight (FHB) is an important fungal disease affecting the yield and quality of barley and other small grains. Developing and deploying resistant barley cultivars is an essential component of an integrated strategy for reducing the adverse effects of FHB. Genetic mapping studies have revealed that resistance to FHB and the accumulation of pathogen-produced mycotoxins are controlled by many quantitative trait loci (QTL) with minor effects and are highly influenced by plant morphological traits and environmental conditions. Some prior studies aimed at mapping FHB resistance have used populations derived from crossing a Swiss landrace Chevron with elite breeding lines/cultivars. Both Chevron and Peatland, a sib-line of Chevron, were used as founders in the University of Minnesota barley breeding program. To understand the native resistance that might be present in the Minnesota breeding materials, a cross of an elite cultivar with a susceptible unadapted genotype is required. Here, a mapping population of 93 recombinant inbred lines (RILs) was developed from a cross between a moderately susceptible elite cultivar 'Rasmusson' and a highly susceptible Japanese landrace PI 383933. This population was evaluated for FHB severity, deoxynivalenol (DON) accumulation and various agromorphological traits. Genotyping of the population was performed with the barley iSelect 9K SNP chip and 1,394 SNPs were used to develop a genetic map. FHB severity and DON accumulation were negatively correlated with plant height (HT) and spike length (SL), and positively correlated with spike density (SD). QTL analysis using composite interval mapping (CIM) identified the largest effect QTL associated with FHB and DON on the centromeric region of chromosome 7H, which was also associated with HT, SL, and SD. A minor FHB QTL and a minor DON QTL were detected on chromosome 6H and chromosome 3H, respectively, and the Rasmusson alleles contributed to resistance. The 3H DON QTL likely represents native resistance in elite germplasm as the marker haplotype of Rasmusson at this QTL is distinct from that of Chevron. This study highlights the relationship between FHB resistance/susceptibility and morphological traits and the need for breeders to account for morphology when developing FHB resistant genotypes.

Project description:BackgroundRice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants.ResultsWe introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants.ConclusionWe demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

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Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter.

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