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Title: Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus

Author
item Hao, Guixia
item Zhang, Shujian
item Stover, Eddie

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2017
Publication Date: 10/19/2017
Citation: Hao, G., Zhang, S., Stover, E.W. 2017. Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus. PLoS One. 12(10):e0186810. https://doi.org/10.1371/journal.pone.0186810.
DOI: https://doi.org/10.1371/journal.pone.0186810

Interpretive Summary: Huanglongbing (HLB or citrus greening disease) and citrus canker are serious threats to the citrus industry. There are no proven strategies to eliminate HLB disease and no commercial cultivars identified with strong HLB resistance. We identified a peptide as one of the most effective antimicrobial peptides. Therefore, we developed tobacco and citrus plants to express the peptide and found that the peptide enhanced disease resistance to canker but not HLB.

Technical Abstract: Citrus Huanglongbing (HLB) associated with ‘Candidatus Liberibacter asiaticus’ (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 containing 23 amino acids was one of the most active AMPs against Xanthomonas citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 in pBinARS/Plus and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation was confirmed by molecular analysis. The level of gene expression in transgenic plants was determined by real-time quantitative PCR (RT-qPCR). We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco and control plants. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants expressing GUS. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by molecular analysis. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with Xanthomonas citri 3213. Transgenic Carrizo plants were challenge for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants nine months after graft inoculation. However, our results showed that transgenic plants expressing D2A21 did not reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Transgenic expression of D2A21 is promising to control citrus canker disease but not HLB. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases.