Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

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.

Establishment of a novel virus‐induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX‐based expression vector

“…Moreover, several research groups have developed transient expression assays using virus‐based expression vectors. In these assays, genes of interest of various pathogens are expressed in N. benthamiana leaves using tobacco rattle virus (TRV) and potato virus X (PVX) expression vectors (Zhang et al, 2019a; Li et al, 2020). Over the past three decades, remarkable progress has been made in the development of plant viral vectors owing to their ease of manipulation (Hefferon, 2017).…”

A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana

“…For example, CLIBASIA_03875 and CLIBASIA_04025 suppress programmed cell death (PCD) (Zhang et al, 2019;Pang et al, 2020), CLIBASIA_05315 targets the chloroplasts and induces cell death in Nicotiana benthamiana (Pitino et al, 2016(Pitino et al, , 2018 as well as physically interacting with the papain-like cysteine proteases (PL) and inhibiting their protease activity in citrus (Clark et al, 2018). In addition, a subset of Sec-dependent secretory proteins were deduced to be candidate effectors that play roles during CLas infection using a Tobacco mosaic virusbased expression system or a strategy for the analysis of temporal and spatial gene expression (Shi et al, 2019;Ying et al, 2019;Li et al, 2020). Recently, two non-classically secreted proteins, CLIBASIA_RS00445 and SC2_gp095, were shown to dramatically downregulate the gene expression of RbohB, a gatekeeper of H 2 O 2 -mediated defense signaling in Nicotiana tabacum (Jain et al, 2015(Jain et al, , 2018, and the former was also identified to suppress oxylipin-mediated defense in citrus plants (Jain et al, 2019).…”

‘Candidatus Liberibacter Asiaticus’ SDE1 Effector Induces Huanglongbing Chlorosis by Downregulating Host DDX3 Gene

“…Liberibacter asiaticus secretes a minimum of 86 proteins via the general secretion system (Sec pathway) [15]. Many of these Sec-delivered effectors (SDEs) have different expression levels in citrus and psyllids to manipulate host responses [16][17][18]. Sec-delivered effector 1 (SDE1, also known as Las5315) is a protein composed of 154 amino acids and can be secreted to the extracellular space outside of the cell through the Sec pathway, upon expression in Escherichia coli [15].…”