| Pepper(Capsicum annuum L.)is an important solanaceous vegetable crop with high nutrition and economic value.However,it is susceptible to Phytophthora capsici infection during the growth process,which seriously affects its production and economic benefits.Therefore,finding genes related to the regulation of pepper blight resistance and studying their regulatory mechanisms are of great significance for pepper resistance breeding.In this study,a differentially expressed gene(Ca SBP05)of pepper SBP-box was screened from the transcriptome databases with the compatible and incompatible Phytophthora capsici based on our lab previous studies.SBP-box genes are a type of plant-specific transcription factor and play important roles in biotic and abiotic stress.However,research on the SBP-box gene is mainly concentrated on model plants such as Arabidopsis and tobacco.Few studies have focused on its role in pepper,especially its defense response to Phytophthora capsici infection.Therefore,in this experiment,based on our previous research,four genes(Ca SBP08,Ca SBP11,Ca SBP12,and Ca SBP13)related to pepper disease were screened from 15 members of the pepper SBP-box gene family.Their functions in pepper in response to Phytophthora capsici infection were identified and the mechanism of regulation was studied.The main results are as follows:1.SBP-box family genes were induced to different degrees,except for the expression of Ca SBP10 was suppressed,under Phytophthora capsici infection.15 members of SBP-box family gene were silenced respectivity,using VIGS.Disease resistance of the detached leaves of the silenced plants was identified.The results showed that the diseased area of detached leaves of Ca SBP08,Ca SBP11,Ca SBP12 and Ca SBP13 gene silenced plants was significantly smaller than that of the control,while the diseased area of the detached leaves of the other gene silenced plants was not significantly different from the control.2.Ca SBP08 protein is located in the nucleus.The expression of Ca SBP08 gene can be induced by salicylic acid and suppressed by its inhibitors.Silencing of Ca SBP08 enhanced pepper resistance to Phytophthora capsici infection.After inoculated with Phytophthora capsici 1d,the expression of defense-related genes(Ca PO1,Ca BPR1,Ca DEF1,and Ca SAR8.2)in Ca SBP08 silenced plants was significantly higher than that of control plants.In the absence of any treatment,the expression of Ca DEF1 and Ca SAR8.2 was increased in Ca SBP08 silenced plants.However,their expression was suppressed in Ca SBP08 transiently expressed plants in pepper.Overexpression of Ca SBP08 in Nicotiana benthamiana increased susceptibility to Phytophthora capsici infection.Overexpression of Ca SBP08 in Arabidopsis can inhibit the expression of upstream(At NDR1,At PAD4,and At TGA4)and downstream genes(At NPR3 and At NPR4)of the salicylic acid signaling pathway.Besides,it induced the expression of downstream(At NPR1)gene of the salicylic acid signaling pathway.In addition,it can induce the expression of At PDF1.2 and At JAR1,which are the downstream genes of the jasmonic acid signaling pathway.Its overexpression in the SID2-2 mutant can induce the expression of At NPR3,At NPR4,At EDS1,At EDS5,At MPK4,At NDR1,At TGA5 and At TGA6 genes in the mutant,and inhibit the expression of At NPR1 and At PR1 genes.Its overexpression in COI1-21 and CO11-22 mutants can inhibit the expression of At PDF1.2 and At PR1 genes in the mutants.In addition,it can also regulate gene expression in salicylic acid and jasmonic acid signaling pathways to varying degrees in co-expressing lines with Na HG.3.Both Ca SBP11 and Ca SBP12 proteins are localized in the nucleus.Ca SBP11 and Ca SBP12 genes were silenced,respectively,which enhanced the defense response of pepper plants to Phytophthora capsici infection.The expression of defense-related genes(Ca PO1,Ca BPR1,Ca DEF1,and Ca SAR8.2)was increased in Ca SBP11 silenced plants 2 days after inoculation with Phytophthora capsici,and was significantly higher than that in control plants.Besides,the expression of these four defense-related genes was significantly higher under incompatible Phytophthora capsici infection than that under compatible Phytophthora capsici infection.Without any treatment,the expression of these four defense-related genes was elevated in Ca SBP11 silenced plants.However,they were inhibited in Ca SBP11 pepper transient expression plants.Regardless of inoculation compatible or incompatible Phytophthora capsici,the expression patterns of these four defense-related genes in Ca SBP12 silenced plants were similar to those in Ca SBP11 silenced plants.Besides,they were also suppressed in Ca SBP12 pepper transient expression plants.The overexpression of Ca SBP11 and Ca SBP12 genes in tobacco,respectivity,reduced the defense response of the transgenic lines to Phytophthora capsici infection.Overexpression of Ca SBP11 in Arabidopsis can induce the expression of salicylic acid signal pathway upstream(At EDS1 and At EDS5)and downstream genes(At NPR1,At TGA5 and At PR1).Inhibits the expression of salicylic acid signal pathway upstream(At PAD4,At SARD1 and At TGA4)and downstream genes(At NPR3 and At NPR4).In addition,it can also inhibit the expression of At PDF1.2 gene,which in the downstream of jasmonate signal pathway.The overexpression of Ca SBP12 in Arabidopsis regulates the expression of At NPR1,At PR1,At NPR3,At NPR4,and At PAD4 as does Ca SBP11 overexpression in Arabidopsis.The difference is that Ca SBP12 inhibits At EDS5 expression and induces At SARD1 expression.Ca SBP11 and Ca SBP12 were over-expressed in SID2-2 mutants,respectivity.It was found that Ca SBP11 can induce the expression of At PR1,At NPR1,At NPR3,At NPR4,At PAD4,At EDS1,At EDS5,At MPK4,At NDR1 and At TGA5,and inhibit At SARD1 and At TGA6 expression in SID2-2 mutant.Except for At PR1,At SARD1,and At TGA6,Ca SBP12 regulates these genes in SID2-2 mutant,as does Ca SBP11 in SID2-2 mutant.Ca SBP11 and Ca SBP12 were overexpressed in COI1-21 and COI1-22 mutants,respectivity.Both Ca SBP11 and Ca SBP12 gene can inhibit At PDF1.2 and induce At PR1 expression in COI1-21 and COI1-22 mutants.4.Ca SBP13 protein is located in the nucleus.Silencing of Ca SBP13 enhanced pepper resistance to Phytophthora capsici infection.The expressions of defense-related genes Ca BPR1 and Ca SAR8.2 were induced in Ca SBP13 silenced plants.However,the expressions of Ca BPR1 and Ca SAR8.2 in Ca SBP13 transient expression plants in pepper were no significantly changed compared with the control.The expression of Ca DEF1 was inhibited in Ca SBP13 transient expression plants in pepper.Overexpression of Ca SBP13 in Nicotiana benthamiana increased susceptibility to Phytophthora capsici infection.5.Under salt stress,Ca SBP12 gene expression was first induced and then suppressed.Ca SBP12 silenced plants enhanced plants,tolerance to salt stress.Under 400 m M Na Cl treatment,the accumulation of reactive oxygen species,the content of malondialdehyde and sodium ions in the target gene silencing plants were smaller than those in the control plants.In addition,without any treatment,the expression of ion transporter genes(Ca SOS1,Ca HKT2-1 and Ca HKT2-2)in the target gene silenced plants was suppressed.Under 200 m M Na Cl treatment,the damage index percentage,sodium ion and malondialdehyde content,active oxygen accumulation,and electrical conductivity of Ca SBP12 over-expressed lines in tobacco were higher than those of wild-type plants,reduced the tolerance of transgenic lines to salt stress. |
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