Identification Of A Susceptibility-Related Gene HW3 For Bacterial Blight Of Rice By Using CRISPR/Cas9 System

Bacterial blight（BB） caused by Xanthomonas oryzae pv. Oryzae（Xoo）, is one of the most destructive diseases of rice（Oryza sativa L.） that could possibly reducing the yield up to 50%. Utilizing host resistance to breed resistant varieties is a worldwide popular way to manage BB for a long time. Up to now, about 42 BB resistant genes（designated in a series from Xa1 to xa41（t）） have been found in rice. However, these resistance genes have varied weaknesses, such as relatively narrow resistance spectrum, weak resistance and recessive inheritance nature. Therefore, continually searching new resistant resources of BB is of great importance for the rice production all over the world. However, use of resistance genes is not the only way to breed BB resistant varieties. DS（disease-susceptibility） gene-based new approach in BB controlling is emerging. Here, we identified a rice DS gene via RNA-seq and pathogenicity assessment of the CRISPR/cas9-targeted mutant plants confirmed that HW3 is a novel DS gene for bacteria blight. The results obtained in this experiment are as follows:1. The RNA-seq data showed that the gene expressed highly in IR24（susceptible line） but lowly in its isogenic-line 5024（resistant line） post inoculation of a strong virulent Xoo strain PXO99 A according to our previous work. The transcriptional level of the HW3 was further investigated via qRT-PCR analyses. The expression profiles were consistent with the transcriptome data. We further reasoned that HW3 might be associated with the BB susceptibility.2. Bioinformatics analysis showed that full length of HW3 gene was 1771 bp with 3 exons and 2 introns. The full length cDNA of HW3 was 1275 bp, encoding a hypothetical protein consisting of 424 amino acids.3. The genomic DNAs and cDNAs of HW3 in 5024 and IR24 were compared carefully by PCR and sequencing, showing that the sequence of their exons and introns were identical. However, a single nucleotide polymorphism was found in their promoters at the 252 site before ATG.4. To verify the subcellular localization of HW3, fusion expressing vector pC1205-YFP-HW3 was constructed and transiently expressed in tobacco epidermal cells. Confocal microscopic observations showed that the YFP-HW3 fusion protein was confined to the nucleus.5. The CRISPR/Cas9 plant expression vector pC-HW3 was constructed according to the sites targeted the first exon of HW3. The Agrobacterium-mediated rice transformation was performed using this CRISPR/Cas9 construct pC-HW3. Totally, 20 T₀ transgenic plants were generated6. All the T₀ transgenic plants were inoculated using leaf-tip clipping method with Xoo strain PXO99 A for the disease symptom evaluation. Obviously, 4 T₀ transgenic plants did not show disease symptom 7 days after inoculation. The lesions length of these 4 resistant plants were shorter compared with IR24 15 days after inoculation, indicating these 4 plants were resistant to infection of PXO99 A.7. We analyzed the types of mutations in the 4 BB resistant and 16 susceptible T₀ plants. We found that all the 4 transgenic plants harbor bi-allelic mutations at target region of HW3, and mutations were detected at both targets. However, the other 16 susceptible T₀ plants do not harbor mutations at target region of HW3. We can conclude that 4 T₀ transgenic plants showed resistant phenotype for the mutated HW3 gene.8. The T₁ plants generated from the 4 resistant plants were inoculated with Xoo strain PXO99 A. We also analyzed the mutation patterns in the T₁ plants. All these different types of mutated plants showed BB resistance phenotype in accordance with T₀ generation. The targeted mutations induced by CRISPR/Cas9 can transmit from T₀ to T₁ generation.9. Seven different homozygous mutant lines of T₂ generation were also inoculated with Xoo strain PXO99 A, showing that lesion length of these homozygous mutant lines were 60⁸0% shorter than IR24. The results validated that the targeted mutations induced by CRISPR/Cas9 can transmit from T₀ to T₁ and T₂ generation and Knockout the DS gene HW3 can enhance the resistance of IR24.