Sweet orange genetic transformation with the attacin A gene under the control of phloem-specific promoters and inoculation with Candidatus Liberibacter asiaticus

ABSTRACT

An alternative approach to control Huanglongbing disease in Citrus is the development of transgenic plants expressing genes that may influence pathogen development. We report herein the production of sweet orange transgenic plants bearing gene constructs containing the antimicrobial gene attacin A (attA)  under the control of phloem-specific promoters. Transcripts of the attA gene were accumulated in the transgenic lines bearing the three gene constructs. However, phloem protein 2 promoters drive higher levels of attA mRNA than sucrose transporter 2 promoter. Candidatus Liberibacter asiaticus (CLas) were transmitted to transgenic plants via infected Asian citrus psyllid or by grafting infected budwood. The efficiency of CLas transmission via infected psyllids was very low and therefore did not allow evaluating the influence of attA expression on bacteria multiplication. On the other hand, CLas transmission via grafting infected budwood was very efficient. Huanglongbing (HLB) symptoms were detected five months after inoculation in both transgenic and non-transgenic inoculated plants. CLas titers, determined 12 months after inoculation, were similar in both transgenic and non-transgenic inoculated plants. However, non-transgenic inoculated plants showed a significant reduction in shoot development comparing to transgenic inoculated plants indicating that the expression of attA gene may influence the plant tolerance to HLB disease.

KEYWORDS:

• Antimicrobial peptide
• bacteria
• Citrus sinensis
• disease resistance
• gene expression
• Huanglongbing

Acknowledgments

The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Núcleo de Pesquisa em Tecnologia e Inovação para Sustentabilidade da Agricultura (NAPTISA/CENA) for research financial support. ECRT acknowledge FAPESP for fellowship and BMJM, FAAMF, JRLS, and MLCV acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research fellowships. The authors thank Fundo de Defesa da Citricultura (FUNDECITRUS) to provide the infected budwood used in the experiments. The first author thanks Dra. Liliane Cristina Liborio Stipp for her support and consulting on gene cloning and elaboration of gene constructs.  The first author also thanks Mateus Santin Mendes, Renata Beatriz Cruz and Marcelo Favareto Correa for their assistance with the experiments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplementary data for this article can be accessed here.

Additional information

Funding

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo [2008/10426-7,2012/21734-0].