Abstract
Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.
Highlights
Huanglongbing (HLB), known as citrus greening, is the most notorious citrus disease worldwide
We reported two ultra-low molecular weight putative virulence factors CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) that caused phloem necrosis in senescent leaves of N. benthamiana when expressing the mature proteins (m03915 and m04250) by tobacco mosaic virus (TMV)
Ca. Liberibacter asiaticus (CaLas) is a phloem restricted uncultured Gram-negative bacterium. It is directly delivered into citrus phloem by the vector of Asian citrus psyllid (ACP) under natural conditions [18]
Summary
Huanglongbing (HLB), known as citrus greening, is the most notorious citrus disease worldwide. The typical symptoms of HLB include yellowing of the veins, dieback of twigs, decline of roots, fruits unable to develop proper color, and, eventually, the death of the infected trees [1,2]. HLB is associated with a phloem-restricted Gram-negative bacterium, named Candidatus Liberibacter africanus (CaLaf), Ca. Liberibacter asiaticus (CaLas), and Ca. L. americanus (CaLam) based on geographic distribution. CaLas is the most virulent pathogen with global distribution [1]. CaLas caused a 72.2% reduction in orange juice and a 20.5% reduction in the fresh fruit market in the United States from 2007–2008 to 2017–2018, respectively [3]. Effective management of HLB is urgently needed. The lack of in-depth knowledge of the virulence mechanism of CaLas prevents the development of effective HLB management
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