Molecular Cloning Flanking Sequences Of T-DNA Integration Of The Ustilaginoidea Virens Mutant Strain B1812,B1241 And Identification Of Proteins Interacting With Uvt-726

Rice false smut disease,caused by Ustilaginoidea virens,is a serious fungal disease occurring in rice panicle.It have posed a serious threat to the quality and yield of rice and endangered human and animal health since the fungus can produce orange smut balls that contaminate rice grain at maturity.Currently,some fungicides have been developed for controlling it basing on biological characteristics,spore germination,isolation techniques and infection mechanism.But this cannot effectively monitor and control the occurrence of false smut and the pathogenic mechanism of U.virens is not very clear.Further study on pathogenic mechanism of U.virens will be useful for controlling false smut and breeding rice resistant varieties.Agrobacterium-mediated transformation technology(ATMT)has become a simple and effective method for genetically modification on pathogens.With wild type strain P1 as a control,biological characteristics,pathogenicity and flanking gene of weak-virulence mutant B1812 and B1241 derived from a T-DNA mutant library by ATMT were analyzed.We would give a preliminary study on function of flanking gene,to provide some theoretical basis for illuminating pathogenic mechanism of U.virens.1.Field inoculation experiments showed that the pathogenicity of B1812 was significantly declined compared with P1.There was no significant difference in colony morphology and growth rate culturing on PSA and TB3.But the growth rate on MM was declined.The conidiation of B1812 was remarkably decreased.The size of conidia was smaller than P1.Amplified HPH after five generations of B1812 inoculated on the PSA without hygromycin indicated that T-DNA had been stably inserted into B1812 genome.Genomic southern blot analysis confirmed that T-DNA was a single copy in mutant B1812.Sequences analysis indicated that 42 bp U.virens sequences were lost in genome of B1812.The full length of UvHac1 which could be translated into transcriptional activator Hac1 was 2196 base pairs,containing a 360 bp of 5’ untranslated regions,a 146 bp of 3’ untranslated regions and a 1690 bp open reading frame encoding 539 amino acids.The T-DNA sequences inserted in the 5’ untranslated regions of UvHac1 and the transcript level of UvHac1 rapidly reduced in the mutant B1812.Haclp involved a bZIP conserved domain,which would play a essential role in regulation of intracellular unfolded protein response.2.There was no significant difference in colony morphology and growth rate between B1241 and P1 culturing on MM,PSA and TB3.Field inoculation trials showed that the pathogenicity of B1241 was significantly declined.After five generations of mutant B1241 inoculated on the PSA without hygromycin,HPH was still amplified,which indicated that T-DNA had been stably inserted into the genome of B1241.Southern blot analysis confirmed that B1241 was a single T-DNA insertional event.Flanking sequence analysis indicated that there were 28 bp loss of U.virens in T-DNA insertion site and 37 bp were not found in T-DNA sequences and U.virens genome.Full length of flanking gene was 2650 bp with a 14 bp of 5’ untranslated regions and a 319 bp of 3’ untranslated regions.The cloned flanking gene UvGH18 was homologous with UV8b-7878 in strain UV-8b.It could encode a glycoside hydrolase family 18 protein which had a DxxDxDxE conserved domain.The T-DNA insertion into the promoter region of UvGH18 led to lower transcription level of UvGH18 in this mutant.We identified four candidates of proteins interacting with GH18 including ubiquitin,putative trehalose-6-phosphate synthase/trehalose phosphatase,putative SUMO ligase SizA and putative cell morphogenesis protein Sog2.3.Uvt-726,a pathogenicity-related gene amplified from weak-virulence T-DNA mutant strain B726,had lower transcriptional level than P1.We construct bait vector by ligating Uvt-726 into plasmid pGBKT7.Toxicity and self-activation of fusion protein were detected.Bait plasmid and library plasmid were transformed into yeast strain Y2H and Y187 respectively.The bait protein and DNA binding domain protein could be coexpressed in yeast diploid and combined with prey proteins in cDNA library of P1,which would activate expression of downstream reporter gene.Yeast diploids were screened by auxotrophic medium DDO/X/A and QDO/X/A.The positive clones were sequenced and would make comparison with whole genomics in U.virens.We identified four candidate proteins interacting with Uvt-726,including AP-3 adaptor complex subunit beta,RNA polymerase II subunit A phosphatase,oligosaccharyl transferase STT3 subunit and ADP/ATP carrier protein.