The Mechanisms Of Histone H2B Monoubiquitination On Regulation Of H₂O₂ Signal During The Defense Responses Against Verticillum Dahliae-toxins In Arabidopsis

Plants usually suffer from various biotic and abiotic stresses during their growth and development.The cotton verticillium wilt,caused by Verticilium dahlia,is a kind of biotic stress.In-depth defense mechanism research is needed to control the verticillium wilt.Previous results show that H2O2 plays an important role in the defense response against Verticillium dahlia-toxins(Vd-toxins)in Arabidopsis.Histone H2B monoubiquitination(H2Bubl)was also involved in this defense response and affects the H2O2 burst.However,the molecular mechanism about H2Bubl in response to Vd-toxins is not very clear.In this study,wild type and H2Bubl loss-of-function mutants hubl-4,hub2-2,hubl-4 hub2-2 were used to further elucidate the role of H2Bubl in regulating H2O2 signal during the defense responses against Vd-toxins in Arabidopsis.After treatment with Vd-toxins,H2Bubl loss-of-function mutants showed sensitive phenotype,including inhibited growth,decreased PCD(programmed cell death)and PR1 expression.These results suggest H2Bubl regulates the defense response against Vd-toxins in a positive way.And then,fluorescence labeling and colorimetry were used to detect H2O2 generation.Compared with the wild type,the production of H2O2 was decreased in the H2Bubl mutants after Vd-toxins treatment,and the rate of H2O2 formation was slower as well.Furthermore,when using real-time PCR to quantitatively detect the expression of RbohD(Respiratory burst oxidase homologue D),the dominant gene involved in H2O2 generation,we found the transcript level of RbohD was suppressed strongly in the H2Bubl mutants.These data indicate that H2Bubl modulates H2O2 production by positively regulating the expression of RbohD in the defence response against Vd-toxins.Western blot experiments showed that MPK3(Mitogen-Activated Protein Kinase 3)and MPK6 were involved in anti-Vd-toxins defense reaction by enhancing kinase activity.The Vd-toxins-induced MPK3 and MPK6 activity enhance was impressed in H2Bubl loss-of-function mutants.It was proved that H2Bubl affected the kinase activity of MPK3 and MPK6 in the defense responses against Vd-toxins in Arabidopsis.Whereafter,we used the mpk3 and mpk6 mutants,overexpression lines of MPK3 and MPK6,and MKK5DD,a mutant that leads to long-lasting aetivation of MPK3 and MPK6,as materials to study the regulation function of MPK3 and MPK6 on H2O2 generation after Vd-toxins treatment.Real-time PCR,fluorescent labeling experiment,DAB and trypan blue tissue dyeing methods were carried out.The results of these experiments showed that after Vd-toxins treatment,mpk3 and mpk6 showed a reduction of H2O2 production,activation of RbohD,and cells death than the wild type.However,the overexpression lines of MPK3 and MPK6,and MKK5DD showed an opposite result.Taken together,MPK3 and MPK6 regulate RbohD transcription and participate in generation of Vd-toxins induced H2O2.The yeast two-hybrid experiment proved that PTP1(Protein Tyrosine Phosphatase 1)and MKP1(MAP Kinase Phosphatase 1)physically interacted with MPK3 and MPK6.PTP1 and MKP1 negatively regulated the kinase activity of MPK3 and MPK6 in arabidopsis.We detected the H2O2 production,activation of RbohD,and cells death in the ptp1,mkp1 mutants and 35S:PTP1,35S:MKP1 after Vd-toxins treatment.The results showed that the H2O2 production,activation of RbohD,and cells death were lower than the wild type in the 35S:PTP1 and 35S:MKP1,and the results were consistent with those in mpk3 and mpk6 mutants.However,ptp1 and mkp1 mutants were the same as wild type,and were consistent with 35S:MPK3,35S:MPK6 and MKK5DD.These data indicate that MPK3 and MPK6 play an important role on regulating H2O2 generation authentically.Yeast single-hybrid experiment and tobacco GUS/LUC enzyme activity detection assay showed that WRKY33 could bind to RbohD promoter region and then promoted the RbohD expression.It is reported that MPK3 and MPK6 could phosphorylate WRKY33 and promote its combination ability with the promoter regions of target genes.There is one possible situation.MPK3 and MPK6 are likely to phosphorylate WRKY33 and promote its combination ability with the promoter regions of RbohD.The combination will influence the RbohD gene expression and H2O2 generation in the defence response against Vd-toxins in Arabidopsis.In conclusion,H2O2 signal is an important signal pathway in the defence response against Vd-toxins in arabidopsis.H2Bub1 directly regulates the expression of the AtRbohD,thereby affecting the generation of H2O2.At the same time,H2Bubl regulates the activity of MPK3 and MPK6,which is possible to regulate the binding activity of WRKY33,indirectly affecting the generation of H2O2.H2Bubl regulates the defence response against Vd-toxins in Arabidopsis by regulating H2O2-mediated signal pathway.From a new perspective,this study reveals the molecular mechanism of H2Bubl in the regulation of H2O2 signaling pathway,which is very important in defence response against Vd-toxins in Arabidopsis.This study also provides theoretical basis for the defense mechanism research against verticillium wilt in plant.