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Article

Identification of the Virulence Factors of Candidatus Liberibacter asiaticus via Heterologous Expression in Nicotiana benthamiana using Tobacco Mosaic Virus

1
Gulf Coast Research and Education Center, University of Florida, Wimauma, FL 33598, USA
2
College of Agronomy and Biotechnology, Southwest University, Beibei, Chongqing 400715, China
3
State Cultivation Base of Crop Stress Biology for Southern Mountainous Land of Southwest University, Beibei, Chongqing 400715, China
4
Heilongjiang Academy of Agricultural Sciences, Harbin 10086, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(22), 5575; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20225575
Received: 15 October 2019 / Revised: 31 October 2019 / Accepted: 4 November 2019 / Published: 8 November 2019
(This article belongs to the Special Issue Host–Pathogen Interaction)
Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide. View Full-Text
Keywords: Huanglongbing; identify virulence factors; growth inhibition; cell death; subcellular localization; protein–protein interaction; virulence mechanism Huanglongbing; identify virulence factors; growth inhibition; cell death; subcellular localization; protein–protein interaction; virulence mechanism
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MDPI and ACS Style

Ying, X.; Wan, M.; Hu, L.; Zhang, J.; Li, H.; Lv, D. Identification of the Virulence Factors of Candidatus Liberibacter asiaticus via Heterologous Expression in Nicotiana benthamiana using Tobacco Mosaic Virus. Int. J. Mol. Sci. 2019, 20, 5575. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20225575

AMA Style

Ying X, Wan M, Hu L, Zhang J, Li H, Lv D. Identification of the Virulence Factors of Candidatus Liberibacter asiaticus via Heterologous Expression in Nicotiana benthamiana using Tobacco Mosaic Virus. International Journal of Molecular Sciences. 2019; 20(22):5575. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20225575

Chicago/Turabian Style

Ying, Xiaobao, Mengyuan Wan, Linshuang Hu, Jinghua Zhang, Hui Li, and Dianqiu Lv. 2019. "Identification of the Virulence Factors of Candidatus Liberibacter asiaticus via Heterologous Expression in Nicotiana benthamiana using Tobacco Mosaic Virus" International Journal of Molecular Sciences 20, no. 22: 5575. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20225575

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