Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes
[Objective]Citrus Huanglongbing(HLB)is a destructive citrus disease primarily caused by Candidatus Liberibacter asiaticus(CLas).The objective of this research is to investigate the growth pattern of CLas in citrus hairy roots at the initial stage of infection,optimize the rapid evaluation system of Agrobacterium rhizogenes-mediated HLB resistance and apply it to the resistance evaluation of antimicrobial peptides.[Method]A plant expression vector,pGNGM 1300,was employed to transform different citrus varieties via the hairy root transformation system,which was mediated by A.rhizogenes K599.Subsequently,varieties with rapid induction and high transformation rate were then selected.Following inoculation with CLas,the presence of CLas in hairy roots was consistently demonstrated through the use of real-time quantitative PCR(qPCR),which allowed for the monitoring of its growth pattern.Further,the optimization of the HLB resistance evaluation system permitted the HLB resistance evaluation of antimicrobial peptides to be conducted via(RT-)qPCR analysis of CLas and related genes,quantification of callose deposition,microstructure observation,and symptom assessment.[Result]Among the 11 tested citrus varieties,citron(Citrus medica)exhibited the fastest induction of hairy roots(15 d),accompanied by the highest induction rate(73.75％)and a substantial transgene positivity rate(53.54％)in hairy roots.The results of regular qPCR monitoring indicated that CLas began to colonize roots on 20-30 days post-inoculation(dpi).At 30-50 dpi,there was a notable increase in the CLas content.The content of the CLas exhibited a slight fluctuation at a range of 60 to 120 dpi,though the discrepancy with 50 dpi was not statistically significant.The results of the antimicrobial peptide resistance evaluation,conducted on MaSAMP(stable antimicrobial peptide),HBD-4(homo sapiens defensin beta 4),and CB(cecropin B),revealed that at 50-120 dpi,the content of CLas in both MaSAMP-and CB-expressing plants was significantly lower than that of the control.However,except that at 90 dpi,the CLas content was also significantly lower in HBD-4-expressing plants than the control.The callose content in all three plants expressing antimicrobial peptides was significantly lower than that of the control(60 dpi).There was no visible cell wall thickening in the phloem,and no significant deposition of starch grains or callus was observed.Furthermore,none of the plants exhibited signs of root death at 90 dpi.[Conclusion]This study elucidates the growth dynamics of CLas in citrus hairy roots during the early stages of infection and optimizes a rapid scoring system for A.rhizogenes-mediated HLB resistance.In addition,the results show that overexpression of MaSAMP,HBD-4 and CB effectively suppresses CLas proliferation,reduces callose deposition and alleviates HLB symptoms.These findings have the potential to be used in the prevention and control of HLB.
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