Genetic Modification Of Potato Late Blight Resistant Genes And Characterization Of The Related Genes For IAA Synthesis In Xanthomonas Oryzae Pv. Oryzae

Potato late blight is a devastating disease which leads to the rot of potato leaf,stem and tuber. Currently, cultivation resistant variety derived from plant resistant gene is the first tactic for control the disaese. In this study, we used three late blight resistance genes, which belong to the same gene family of CC-NBS-LRR type, shared with about 82% identities with amino acid sequence. Both Rpi-mcq1.1 and Rpi-mcq1.2 were cloned from Solanum mochiquense, and Rpi-vnt1.1 was cloned from Solanum venturii . By changed with Rpi-vnt1.1 gene promoter, domain swapping of the Rpi-mcq1.1 and Rpi-mcq1.2, we goal to seek after resistance improvement and to explore the domain that controlled the recognization between R gene and pathogen effector.Firstly, three new construts have been made with overlapping PCR technique and domain swap tactic. The V1M2 was combinated with CC-NB motif of Rpi-vnt1.1and LRR motif of Rpi-mcq1.1. RpiV::mcq1.1 was made by replaced the native promoter with Rpi-vnt1.1 promoter . RpiV::mcq1.2 was made by replaced with Rpi-vnt1.1 promoter as RpiV::mcq1.1.Three new genes were ligated into pCAMBIA1300, and mobilised into Agrobacterium strain Aglâ… , then introduced into suceptible potato variety Desiree and Nicotiana benthamiana. For each construct V1M2, RpiV::mcq1.1 and RpiV::mcq1.2, 13, 11 and 25 positive individuals were conducted with potato Desiree regarded as 73%, 61% and 79% of transformation efficiency respectively. The amout of 8, 7 and 13 positive individuals were conducted in N. benthamiana with 37%, 30% and 41% of transformation efficiency respectively. All the materials will be tested for inoculation Phytophthora infestans, to test whether their resistant spectrum have changed or not, and to detect whether they have been affected to recognize the Avr2.By using Agrobacterium mediated transient expression system on N. benthamiana, Rpi-mcq1.1, V1M2, RpiV::mcq1.1 and RpiV::mcq1.2 were indicated to recognize the Avr2 (G3, G12) after co-infiltration with GV3101 carried each resistance gene and Avr2. The results support to make aconclusion that it is LRR motif of Rpi-mcq1.1 correspond for the recognization but not the CC-NBS motif. More interestingly, we found that Rpi-mcq1.2 was turns to recognize to Avr2 after replaced with Rpi-vnt1.1 promoter which couldn't trigger HR on N. benthamiana after co-infiltration with it original gene and Avr2. Together with the results that Rpi-mcq1.2 can recognize to Avr2 after dedrived with 35S promoter, we suggest that the interaction between Rpi-mcq1.2 and Avr2 is a weakly interaction, maybe the dosage-dependent reaction. This is basis for analysising interaction betwern Rpi-mcq1.1, Rpi-mcq1.2, Rpi-vnt1.1 LRR domain and avirulence gene Avr2.Meanwhile, we surprise to found that Rpi-vnt1.1 can auto-activate the HR on response in N. benthamiana. In order to explore the function motif response for this function, we constucted a series of vector as below which included pCAMBIA1300+Rpi-vnt1.1 promoter -CC-NB, pBin19s+35 promoter+ Rpi-vnt1.1CC-NBS, pBin19s+35s promoter + Rpi-vnt1.1 LRR, pBin19s+35s promoter+ Rpi-vnt1.1 CC-NBS-LRR, pBin19s+35s promoter+Rpi-mcq1.1 CC-NBS, pBin19s+35s promoter+ Rpi-mcq1.1LRR and pBin19s+35s promoter+ Rpi-mcq1.1 CC-NBS-LRR. After N. benthamiana transient expression assay, still only Rpi-vnt1.1 with its native promoter has the property. The results indicated that the promoter and LRR motif was invovled in this self auto-activation. Those resluts will provide to further studies.Auxin is an essential plant hormone. Auxin regulates plant growth and development. The main activity of the natural form of auxin is indole-3-acetic acid, IAA. Rice bacterial blight disease caused by Xanthomonas oryza pv. oryza,is the most devastating diseases of rice worldwide. This diseases cause tremendous yield loss each year. Recent studies have shown that IAA not only regulate the growth and development of rice, also as virulence factors when bacterial blight and bacterial infection.Preliminary study have shown Xanthomonas oryza pv. oryza can produce IAA. There may be have a new synthesis pathway of IAA in plant pathogenic bacteria. The purpose of this research is the built of mutant from the method of homologous recombination. Identification the pathway of IAA biosynthesis in Xoo may help us text the function of IAA in the process of pathogen infect rice. Our study may have deep meaning in pathogenesis of Xoo.According to IAA biosynthesis pathway related the amino acid sequence databases of Arabidopsis thaliana in the NCBI BlastP, found the genome Xoo PXO99 homologous gene PXO₀0867 (Nitric Oxide Hydrolase Gene Family). We construction PXO99 mutation via homologous recombination PXO ₀0867. Using PR-PCR and gene sequencing method to verificate PXO99 mutant strain, confirmed the accuracy of strain genotypy.We extracted IAA with ethyl acetate . By high performance liquid chromatography ,we texted the IAA content of metabolites in the wild-type PXO99 and PXO99â–³PXO₀0867 . The results showed the IAA content of PXO99â–³PXO₀0867 is slightly less than PXO99. It proved PXO₀0867 gene have effect in PXO99, but not play an important role in IAA biosynthesis pathway .we take transgene Dr5-GUS rice leaves as the experimental material, inoculated PXO99 and PXO99â–³PXO₀0867. By GUS staining method and microscope,we examine the stain of rice leaves. We found PXO99â–³PXO₀0867 infected leaves infected have lighter color than PXO99. This result consistent with PXO99â–³PXO₀0867 has lower-level produced of IAA.Multifunctional enzyme analysis the transgene Dr5-GUS rice leaves that inoculated PXO99 and PXO99â–³PXO₀0867. The results showed PXO99â–³PXO₀0867 has lower stimulate value than PXO99. Consistent with the expected results.While PXO99â–³PXO₀0867 and PXO99 inoculated rice leaves of different varieties to rice lesion analysis. The experimental results, the rice has slight disease course when infected PXO99â–³PXO₀0867 the initial description of IAA in the process of pathogen infection plays a role.In summary, we have constructed PXO99 mutantion by homologous recombination method. This method plays a guiding role for other genes. Meanwhile the result of this study play a role in finding the action of IAA produced by Xoo when Xoo infect rice, and it is helpful for us finding the pathogenesis of Xoo.