Expression And Functional Evaluation Of CabZIP53 And CaZNF830in Pepper Defense Against Bacterial Wilt Disease And High Temperature-high Humidity

Pepper(Capsicum annuum)is one of the most important vegetable worldwide,diseases such as blight and bacterial wilt caused by PC and RS frequently lead to heavy loss in pepper production,especially under the condition of high temperature and high humidity(HTHH).Dissection of molecular mechanism underlying pepper immunity will benefit its genetic improvement,which is the most efficient way to solve the problem caused by pepper diseases.Since plant immunity against different pathogens are largely regulated at transcriptional level with the action of different transcription factors.The functional characterization of TFs involved in plant immunity is a feasible approach for the elucidation of plant immunity.However,the roles of different transcription factors in pepper immunity remain largely elusive.In the present study,the functions of two pepper transcription factors,CabZIP53 and CaZNF830,were functionally characterized.The main results are as follow:1.A putative bZIP transcription factor(CA04g07280)was focused.Since a subset of pathogen responsive cis-elements were found to be present in its promoters and it was found to be the possible target of CaWRKY40,a regulator of pepper response to RSI confirmed by our previous study.Its deduced amino acid sequence contains a highly conserved bZIP domain and exhibits the highest identity to that of StbZIP53 among all members of the bZIP family in tomato,and was designated as CabZIP53.2.CabZIP53 was up-regulated in pepper plants upon Ralstonia solanacearum inoculation(RSI)or HTHH treatment.The silencing of CabZIP53 by virus-induced gene silencing(VIGS)significantly enhanced susceptibility of pepper plants to RSI.The recorded data clearly manifested an increasing trend of disease index and pathogen growth in CabZIP53-silenced plants.Clear wilting phenotype appeared in TRV2::CabZIP53 plants while TRV2::00plants did not display obvious wilting signs,accompanied with down regulated transcription of the tested immunity marker genes including CaPR1,CaNPRl,CaHIRl and CaABRl.Additionally,TRV2::CabZIP53 plants,when subjected to high temperature(38℃)and 90%humidity,showed significantly low thermo-tolerance as compared to control plants.The expression of thermo-tolerance related gene i.e.CaHSP24 was much lower in TRV2::CabZIP53 in comparison with TRV2::00plants.Taken together,these findings demonstrate that CabZIP53 silencing significantly compromised basal defense/tolerance of pepper plants to RSI or HTHH.3.By transient over-expression in pepper plants,the over-expression of CabZIP53 triggered significant HR mimic cell death,accumulation of H2O2 and immune reaction,manifested by darker trypan blue and DAB staining as well as the upregulation of the tested immunity or thermo-tolerance associated marker genes including CaPRl,CaNPRl,CaHIRl and CaABR1or CaHSP24.4.We also evaluated relationship between CaWRKY40 and CabZIP53in their expression,our data showed that they relate with each forming feedback loop,the transient over-expression of CaWRKY40 act the transcription of CabZIP53,while the transient over-expression of CabZIP53 also up-regulated the expression of CaWRKY40.In addition,the promoter of CabZIP53 was found to be directly bound by CaWRKY40 by ChIP-PCR.5.As Zinc finger(ZNF)transcription factors regulate important processes in plants such as development,vegetative activities and plant immunity.Despite their immense significance,majority of ZNF transcription factors(TF)involved in pepper immunity and resistance to heat stress have not been focused much.Herein,we identified and functionally characterized a putative ZNF(CA12g02120)in pepper defense against Ralstonia solanacearum inoculation(RSI)and tolerance to high temperature and high humidity(HTHH).This putative ZNF was focused because its promoter contains several pathogen or heat stress responsive cis-elements,its deduced amino acid sequence shares 87%,85%,85%,82%,61%and 45%identity in amino acid sequence to ZNF proteins in other plant species,and was designated as CaZNF830.Consistent with the presence of pathogen responsive cis-elements in its promoters,CaZNF830 was found to be regulated by RSI or HTHH in pepper plants.Activity of GUS driven by pCaZNF830 was significantly enhanced by RSI and HTHH in comparison with the control plants implying its possible roles in pepper immunity against RSI or HTHH.6.The silencing of CaZNF830 by virus induced gene silencing(VIGS)significantly compromised pepper immunity against RSI with enhanced growth of Ralstoniasolanacearum in pepper plants.Silencing of CaZNF830 also impaired tolerance to HTHH coupled with decreased expression levels of immunity and thermo-tolerance associated marker genes including CaHIRl,CaNPR1,CaPR1,CaABRl and CaHSP24.7.The transient over-expression of CaZNF830 in pepper leaves by infiltration of GV3101 cells containing 35S::CaZNF830-HA induced HR mimic cell death,H2O2 accumulation and activated the transcriptions of the tested defense-relative or thermo-tolerance associated marker genes.RT-PCR and immune-blotting assay confirmed the stable expression of HA-tagged CaZNF830 mRNA and protein in pepper.All these results suggest thatCabZIP53 and CaZNF830 acts as positive regulators of plant immunity against RSI or tolerance to HTHH.These results indicate a tight linking between pepper response to RSI and HTHH,which may be the result of the evolutionary adaptation of simultaneous Ralstonia infestation and high temperature and humidity stress.The results of this paper not only benefit the dissection of pepper immunity against pathogens but also provide insight into genetic improvement of plants for greater productivity.