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

Abstract

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.

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

“…Based on the genomic information of CaLas, several reports focused on the virulence mechanisms of CaLas [7][8][9][10]. The identified effectors could cause cell death of infiltrated leaves [6,8], systemic leaves necrosis [7], and inhibition of plant growth [8]. Mature proteins of CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) caused phloem necrosis in the senescent leaves of N. benthamiana when expressed by TMV ( Figure 2) and functioned at the protein level (Figure 3).…”

“…This finding indicated that CLIBASIA_03915 and CLIBASIA_04250 were Sec-Dependent proteins. TMV was used to express virulence factors of CaLas and resulted in identifying two virulence factors that inhibited plant growth at two weeks post infiltration (WPI) [8]. Heterologous expression of m03915 and m04250 in N. benthamiana by TMV showed a similar phenotype to control GFP at 2 WPI.…”

“…The effectors of CaLas mainly rely on Sec secretion machinery to enter host cells. Several reports have used transient expression or viral vectors to identify the effectors of CaLas [6][7][8]. CaL5315 was reported as a lethal effector, causing cell death in infiltrated N. benthamiana leaves by transient expression [6].…”

“…CLIBASIA_00460 caused necrosis in the systemic leaves when expressed by Potato virus X (PVX) in N. benthamiana [7]. CLIBASIA_00470 and CLIBASIA_04025 were reported to inhibit N. benthamiana growth when expressed by tobacco mosaic virus (TMV) at two weeks post infiltration (WPI) [8].…”

“…The mature sieve elements contain smooth endoplasmic reticulum (ER), mitochondria, sieve element plastids, and phloem proteins [15]. It was recently reported that CaLas encodes proteins to interact with the key proteins of lignin production [8], which is the main component of woody cell wall biomass (up to 30%) [16].…”