Molecular Mechanism Study Of Xanthomonas Oryzae Pv.oryzae TAL Effectors Transcription Activation Of Host Genes

The Xanthomonas genus infect a broad range of crops.It severely impacts the yield quantity and quality of important crops by causing symptoms with leaf blights,streaks,spots,stripes,necrosis,wilt,cankers and gummosis on leaves,stems and fruits in a wide variety of plants.Xanthomonas inject a plethora of diverse virulent factors into the host cell during infection.Xanthomonas oryzae pv.oryzae(Xoo)is Gram-negative plant-pathogenic bacteria,which is the major rice disease bacteria.Transcription activator-like effectors(TALEs)injected by Xoo through type III secretion system could hijack y subunit of host transcription factor IIA(TFIIAy)to activate the transcription of host susceptibility gene(S gene).The molecular mechanism of the interaction between TALEs and TFIIAy remains poorly understood.Functional TFIIA is a ternary complex comprising α,β and γ subunits.However,whether TALEs recruit TFIIAα.TFIIAβ,or both remains unknown.The molecular mechanisms by which TALEs mediate host susceptibility gene activation require full elucidation.Here,we show that TALEs are unable to recruit holo-OsTFIIA(α+β+γ/αβ+γ)three subunits.In sharp contrast,TALEs interact with TFIIA(a+y)binary subcomplex to reconstitute TALEs+a+y complex.The transcription factor binding(TFB)regions of TALEs have a dominant role in these interactions.We identified that TFB region which recruited TFIIA(a+y)binary subcomplex contains 72 amino acids(residues 1044-1115 of PthXo1 effector).The reconstituted TFB+a+y complex interacted with TATA-box binding protein(TBP)as TFIIA(α+β+γ)did in vitro.Furthermore,the interaction between TALEs and the a+y complex exhibits robust DNA binding activity in vitro.Based on these results,we propose a new model for TALE-mediated transcriptional activation of downstream susceptibility genes.By mimicking the function of the basal TFIIAβ subunit,TALEs successfully recruit other transcription factors to assemble the preinitiation complex to activate the transcription of host disease susceptibility genes.Sequences alignment showed that TFB regions are highly conserved in Xanthomonas species.We also found that TFIIAys in plants are highly conserved.We speculate that our new mechanism is general among plant species.We devoted to determining the crystal structure of TFB+TFIIAα+TFIIAγ complex or TFB+TFIIAγ complex to uncover the mechanism of their interaction.After 400 clones’construction and 520 protein expression tests,we successfully purified the TFB+TFIIAα+TFIIAγ complex and TFB+TFIIAγ complex.TFB+TFIIAγ complex was in bad behavior and aggregated in SEC(size exclusion chromatography).It was easy to get insoluble.In contrast,TFB+TFIIAα+TFIIAγ complex was in good behavior and not aggregated.We screened about 20000 crystallize buffer but did not get any crystals.We identified some residues in TFIIAγ that may associate with the interaction between TFB and TFIIA(α+γ)through mutations and SEC assays.We determine the crystal structure of rice holo-TFIIA(α+β+γ).This is the first structure of plant TFIIA.The structure of rice TFIIA is highly conserved with that of yeast and human.It will provide clues for determining the structure of TFB+α+γ.Above all,the uncovered mechanism widens new insights on host-microbe interaction and provide an applicable strategy to breed high-resistance crop varieties.