Function Of CsSAMT-1 In Salicylic Acid Signal Response To Citrus Huanglongbing Infection

Huanglongbing?HLB?is the most destructive citrus disease in the world.The most causal agent of HLB in the world is‘Candidatus Liberibacter asiaticus'?CLas?,which living in the citrus phloem.Plant defense against pathogen attack need activate both locally acquired resistance?LAR?and systemic acquired resistance?SAR?.Salicylic acid?SA?and methyl salicylate?MeSA?play a central role in this process.Salicylic acid carboxyl methyltransferase?SAMT?can convert SA to MeSA,which plays an important role in translocation of SAR signaling from infected tissue to systemic tissue through the phloem.In order to investigate the intrinsic mechanism of salicylic acid signal pathway regulating citrus HLB tolerance,we compared the the differences in host tolerance from symptoms,pathogen growth,changes of host anatomical structure and level of SA and MeSA between sour pomelo and Jincheng orange.Based on the closely connection of MeSA and HLB tolerance,we studied the key gene CsSAMT-1 of MeSA signal.The principal research results are as follows:1.Tolerance analysis of sour pomelo and Jincheng orange against HLB.The resistance evaluation showed that the time for sour pomelo to be infected byHLB was longer than that of Jincheng orange..The symptom of sour pomelo after infection was milder than that of Jincheng orange.There is little accumulation of starch granules and callose in sour pomelo,which contains more pathogens and slightly thickened phloem.The results showed that sour pomelo has tolerance to HLB compared with Jincheng orange.2.The difference of SA and MeSA level in sour pomelo and Jincheng orange.The HPLC-MS/MS determination showed that the contents of SA and MeSA in sour pomelo midrib were 9.02 ng·g^-1?FW?and 12.33 ng·g^-1?FW?respectively,which were significantly higher than those in Jincheng(1.86 ng·g^-11 and 0.04 ng·g^-1,FW)in control test.After HLB infection,the level of SA and MeSA in Jincheng orange increased,especially in the midrib increased by 10.58 times(19.69 ng·g^-1,FW).In contrast,the SA in the midrib of infected sour pomelo decreased to 3.74 ng·g^-1?FW?,and MeSA increased to 16.02 ng·g^-1?FW?.In infected Jincheng orange,MeSA was still far less than that in the infected sour pomelo.The results showed that the levels of SA and MeSA in the sour pomelo were significantly higher than that in Jincheng orange.It is speculated that the relatively high tolerance of sour pomelo to HLB may related to high accumulation of MeSA.3.Differences of CsSAMT-1 gene expression between sour pomelo and Jincheng orange.qRT-PCR analysis showed that the expression level of CsSAMT-1 in the infected Jincheng orange was significantly increased compared with the control plants,and its expression level in the midrib was significantly higher than that in the mesophyll.The expression of CsSAMT-1 in mesophyll and midrib of sour pomelo control was respectively 194.56 times and 57.16 times that of Jincheng orange.After HLB infection,it decreased to 4.08 times and 5.48 times,but was still higher than that of Jincheng.The results showed that the CsSAMT-1 expression was induced by HLB in Jincheng orange,but expressed relatively high in sour pomelo before HLB infection.4.Expression analysis of CsSAMT-1 induced by SA and MeSA.Both SA and MeSA induced the expression of CsSAMT-1 gene.The highest expression level of CsSAMT-1 was detected at 12 h and 6h after induction in Jincheng orange and in sour pomelo.The results showed that CsSAMT-1 responses earlier to SA and MeSA in sour pomelo compared with Jincheng orange.5.Cloning and informatics analysis of CsSAMT-1 gene.The CsSAMT-1 gene was cloned from HLB infected sour pomelo and Jincheng.The amplified sequence of CsSAMT-1 contains a 1113 bp open reading frame,which encodes 370 amino acid residues.There is a typical conserved domain of Methyltransf₇ methyltransferase between the 42nd to the 370th amino acids.SAM is the methyl donor in the conversion reaction,and salicylic acid is one of the substrates.Its primary structure is a hydrophilic stable protein,which is more homologous to SAMT of cocoa and cotton according to evolutionary analysis.6.Overexpression and RNAi-repression of CsSAMT-1 in Jincheng orange.Under the control of a strong CaMV35S promoter,the plant expression vector pLGNCsSAMT-1 and pUCRNAiCsSAMT-1 were constructed and then transformed Jincheng orange by Agrobacterium tumefaciens-mediated transformation.After GUS staining and PCR identification,sixteen transgenic oranges overexpressing CsSAMT-1and four RNAi-interfering transgenic plants were obtained.Compared with wildtype,over-expressed transgenic plants have more round leaf shape,deeper green leaf color,and thicker leaves at the early stage of grafting seedlings.However,the difference in leaf color and leaf shape disappeared as the plants grew.Most of the transgenic plants had no significant differences in leaf length and leaf width,while almost all transgenic plants had significantly thicker leaves compared with wildtype plants7.Analysis of gene expression and hormone levels in transgenic Jincheng orange.For transgenic plants over-expressing CsSAMT-1,the CsSAMT-1 expression level,SA and MeSA content were significantly higher than the control.The expression level of CsSAMT-1 was significantly decreased in RNAi-interfering transgenic plants,but the level of SA and MeSA was significantly increased.It demonstrated that CsSAMT-1catalyzes the conversion of SA to MeSA.The above data indicated that CsSAMT-1 plays an important role in MeSA response to HLB.It is possible to manipulate CsSAMT-1 to enhance tolerance of HLB through accelerating the conversion of SA to MeSA in citrus.