Functional Characterization Of CaZhp2 And CaHsfB2a In Pepper Response To Ralstonia Solanacearum Infection Or High Temperature And High Humidity Exposure

Pepper(Capsicum annuum)is an important vegetable and industrial raw material crop.Due to its high production efficiency,the planting area of pepper has been increasing in recent years.The diseases caused by its accompanied associated pathogens in the soil under high temperature and high humidity often affect the yield and quality of pepper,resulting in a decrease in their production efficiency.Development and application of disease-resistant pepper cultivars is an important countermeasure to solve problem caused by pepper diseases during pepper production.The elucidation of the molecular mechanisms of pepper resistance to diseases and high temperature and high humidity tolerance is an important basis for the effective genetic improvement of pepper disease resistance.A large number of studies have shown that plant disease resistance is mainly regulated at the transcription level by with the action of various transcription factors.Therefore,analysis and elucidation of transcription factor-mediated transcription rearrangement is an important way to elucidate the resistance mechanism of pepper.In this study,we performed a genome-wide analysis of the zinc finger homeodomain(ZFHD)and heat shock factor(HSF),and studied the possible role of CaZhp2 and CaHsfB2a in pepper resistance to bacterial wilt or high temperature tolerance.The main results are as followings:1.Through promoter analysis,we identified pepper CaZhp2 and CaHsfB2a genes have potential cis-acting elements in their promoters for immunity tolerance.The results showed that immunity associated ciselements in promoters of pCaZhp2 and pCaHsfB2a are TGACG-motif,HSE,GARE,TC-rich repeats,W-box and TGACG-motif,ABRE,ERE,TC-rich repeats,G-box,W-box,respectively.2.Based on the distribution analysis of cis-acting elements in their promoters,the response of ZFHD family member CaZhp2 was identified for further functional characterization.Its response to R.solanacearum inoculation or high-temperature and high-humidity treatment was determined,the results showed that CaZhp2 was upregulted by R.solanacearum inoculation or by HTHH exposure as well as by exogenously applied SA,MeJA,ETH and ABA.Subcellular localization analysis revealed that the fusion protein CaZhp2-GFP was localized exclusively in the nuclei,whereas the control GFP was distributed throughout the cell;the virus-induced silencing of CaZhp2 resulted in a significant reduction in the level of resistance to R.solanacearum in pepper plants,accompanied by downregulation of CaHIRl,CaABR1,CaWRKY40,and CaNPR1 and increased growth of R.solanacearum;virus-induced CaZhp2 silencing pepper plants also exhibited higher sensitivity to high temperature and humidity stress relative to control and downregulation of high temperaturerelated gene CaHSP24 expression;Agrobacterium mediated CaZhp2 transient overexpression resulted in the appearance of HR mimic cell death and H2O2 accumulation in pepper leaves near the injection sites,manifested by deeper trypan blue and DAB staining and higher leaf electrolyte leakage,accompanied by upregulation of CaHIRl,CaABR1,CaWRKY40,CaNPR1,and CaHSP24.In addition,we also found that the transient overexpression of CaZhp2 led to upregulation of CaWRKY6,CaWRKY27 and CaWRKY40,and downregulation of CaWRKY58.These results indicate that CaZhp2 is upregulated by RSI or HTHH in pepper plants thus directly or indirectly increase the expression of disease-resistance or high-temperature-resistancerelated genes to increase the level of resistance against bacterial wilt or high temperature tolerance in pepper plants.Furthermore,CaZhp2 may be associated with CaWRKY6,CaWRKY27,CaWRKY40,and CaWRKY58 in expression and function,forming a transcriptional regulatory network that plays a role in heat tolerance or disease resistance in pepper plants.3.Also based on the distribution analysis of cis-acting elements,CaHsfB2a was identified from the HSF family to further functionally characterize in pepper response to high temperature and high humidity exposure or R.solanacearum infection.The results showed that CaHsfB2a was up-regulated by high temperature and high humidity exposure or R.solanacearum inoculation.In addition,CaHsfB2a was also upregulated by exogenous application of SA,MeJA,ETH and ABA from 1 to 24 hpt.Subcellular localization assay revealed that the CaHsfB2a-GFP fusion protein targeted exclusively in the nuclei,while the control GFP was distributed throughout the cell;the silencing of CaHsfB2a by virus induced gene silencing(VIGS)resulted in a significant decrease in the resistance of the pepper plants to high temperature and high humidity exposure or resistance to R.solanacearum inoculation,accompanied with downregulation of disease resistance or high temperature tolerance associated marker genes including CaHIR1,CaABR1,CaWRKY40,CaNPRl and CaHSP24,accelerated growth of Ralstonia solanacearum in pepper plants.A Agrobacterium-mediated transient overexpression of CaHsfB2a was performed in pepper leaves,it was demonstrated that transient overexpression of CaHsfB2a significantly upregulated the expression of the above-mentioned immunity or high temperature tolerance associated marker genes.Furthermore,the transient expression of CaHsfB2a significantly increased the expression of CaWRKY6,CaWRKY27,and CaWRKY40,which are positive regulators in pepper resistance toward Ralstonia solanacearum,and decreased CaWRKY58,a negative regulator in pepper immunity against Ralstonia solanacearum.These results indicate that CaHsfB2a is upregulated during the infection of pepper by Ralstonia solanacearum or challenge of pepper plants by high temperature and high humidity stress,therefore enhances the tolerance to high temperature and high humidity and resistance to Ralstonia solanacearum by transcriptional modulation of immunity or heat stress tolerance associated genes,forming transcriptional networks with TFs including CaWRKY6,CaWRKY27,CaWRKY40 and CaWRKY58.The results in the present study indicate that both CaZhp2 and CaHsfB2a play positive regulatory roles in response of pepper to hightemperature and high-humidity stress and R.solanacearum infection,forming transcriptional regulatory networks with CaWRKY6,CaWRKY27,CaWRKY40 and CaWRKY58 to fine tune the defense reaction against these stresses.These results indicate that the high temperature tolerance and bacterial wilt resistance in pepper are closely related,which may be resulted from the natural selection pressure from the enhanced growth,development and virulence of pathogens such as Ralstonia solanacearum under condition of high temperature and high humidity.However,the hierarchical structure of the transcriptional regulatory network composed of CaHsfB2a,ZFHD and CaWRKY6,CaWRKY27,CaWRKY40 and CaWRKY58 and their mechanisms of action remain to be elucidated.