Interaction of Liberibacter Solanacearum with Host Psyllid Vitellogenin and Its Association with Autophagy

Abstract

Candidatus Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major constraint for carrot production in Israel. Unveiling the molecular interactions between the psyllid vector and CLso can facilitate the development of nonchemical approaches for controlling the disease caused by CLso. Bacterial surface proteins are often known to be involved in adhesion and virulence; however, interactions of CLso with carrot psyllid proteins that have a role in the transmission process has remained unexplored. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast two-hybrid system assay. We identified psyllid vitellogenin (Vg) to interact with both OmpA and flagellin of CLso. As Vg and autophagy are often tightly linked, we also studied the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy. Induced Vg expression significantly suppressed autophagy-related gene expression and the levels of CLso significantly increased, resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter. IMPORTANCE Pathogen transmission by vectors involves multiple levels of interactions, and for the transmission of liberibacter species by psyllid vectors, much of these interactions are yet to be explored. Candidatus Liberibacter solanacearum (CLso) haplotype D inflicts severe economic losses to the carrot industry. Understanding the specific interactions at different stages of infection is hence fundamental and could lead to the development of better management strategies to disrupt the transmission of the bacteria to new host plants. Here, we show that two liberibacter membrane proteins interact with psyllid vitellogenin and also induce autophagy. Altering vitellogenin expression directly influences autophagy and CLso abundance in the psyllid vector. Although the exact mechanism underlying this interaction remains unclear, this study highlights the importance of immune responses in the transmission of this disease agent.

Keywords: Liberibacter; autophagy; outer membrane proteins; psyllid; vitellogenin.

FIG 1

Interaction between Liberibacter membrane proteins and host vitellogenin. (A) Yeast two-hybrid assay showing strong interaction between Vg-VWD domain and bacterial OmpA (1a, 1b) and Flagellin (2a, 2b) in QDO+Xgal (a) and QDO+Xgal+Aba plates (b). Subsets 3a and 3b show negative control showing no interaction between empty pGADT7 vectors with bacterial proteins. (B) and (C) Detection of Vg-VWD with N-terminal His-tag after pulldown assay using GST-tagged OmpA (B) and Flg (C) as baits by SDS-PAGE (a) and Western blot (b) using anti-His antibody. (D) Immunostaining of Ca. L. solanacearum+ midguts with anti-Vg antibody (green) and anti-Ca. L. solanacearum antibody (red) showing spatial colocalization of the two (yellow) under confocal microscopy.

FIG 2

Structure and domain architecture of vitellogenin. (A) Vg contains three conserved domains; Lipoprotein LPD_N-terminal domain, DUF1943, and VWD domain. (B) Three-dimensional structure of psyllid Vg, modeled by iTasser with highest C-score of 0.32 shows the two major domains: LPD_N (red) and VWD (blue). (C) Phylogeny of amino acid sequences of all known psyllid Vg proteins showing clustering within Hemipteran clade with T. evansi used as an outgroup.

FIG 3

Expression profiles of vitellogenin (Vg) and the autophagy-related (Atg) genes CaspaseI, Cathepsin B, Atg16, Atg2, and Atg5 in Ca. L. solanacearum uninfected and infected psyllids. (A) Relative expression using real-time PCR showing upregulated gene expression of Vg and Atg-genes in Ca. L. solanacearum+ whole bodies and midguts compared to Ca. L. solanacearum-psyllids (*, P < 0.05; ***, P < 0.001). (B) Higher expression of Vg (green) in Ca. L. solanacearum-infected midguts compared with Ca. L. solanacearum-free as seen using immunostaining. (C, D) Staining of acidic compartments (lysosomes and autolysosomes) using LysoTracker Green showing higher lysosomal activity in Ca. L. solanacearum+ midguts (C) and in ovaries (D) compared with Ca. L. solanacearum-psyllids.

FIG 4

Effect of JH-III hormone on Vg and Ca. L. solanacearum. (A) Relative expression of Vg in male and female whole body, midguts, and in ovaries showing induced expression of Vg throughout with females showing much higher expression than in males, upon JHIII application than in control (L+C) (*, P < 0.05; ***, P < 0.001). (B) Relative titer of Ca. L. solanacearum (Omp) in female whole bodies, midguts, and in ovaries after JH-III application (P ≤ 0.05). (C) Elevated Ca. L. solanacearum titer in the hemolymph of JH-III treated psyllids (*, P < 0.05; ***, P < 0.001). The fold change for each gene is mentioned beside the bars. (D, E) Immunostaining of Vg and Ca. L. solanacearum showing induction of Vg (green) expression and increase in Ca. L. solanacearum titer (red) upon JH-III application along with their colocalization. The colocalization was validated using ImageJ with Pearson’s correlation coefficient (R value) of 0.75 and 0.89 for guts and ovaries, respectively.

FIG 5

Effect of JH-III on autophagy. Relative expression of lysosomal and autophagy genes in whole bodies (A), midguts (B), and ovaries (C) showing downregulation of all the known genes upon JH-III application (P ≤ 0.05). (D, E) Representative images showing reduction of autophagy and lysosomes in the JH-III applied psyllids. Staining of the midguts (D) and ovaries (E) with DAPI (blue) and lysosomes (green) with b and d showing magnified images of the insets in a and c, respectively. *, P < 0.05; ***, P < 0.01.

FIG 6

Effect of Thapsigargin and Rapamycin on Vg, Ca. L. solanacearum, and autophagy. (A) Relative gene expression of Vg, autophagy genes and Ca. L. solanacearum titer in the psyllid midguts upon Thapsigargin application (P < 0.05). (B) Relative gene expression of Vg, autophagy genes, and Ca. L. solanacearum titer in the psyllid midguts upon Rapamycin application (P < 0.05). (C) Staining of lysosomes (green) and nuclei (blue) showing disintegrated nuclei and absence of autophagy upon Thapsigargin application (b), and increase in lysosomal activity upon Rapamycin application (c) compared with the control midguts (a). Lower panel showing decrease in vitellogenin and increase in Ca. L. solanacearum titer upon Thapsigargin application (e) and lower Ca. L. solanacearum abundance upon Rapamycin application (f) compared with control midguts (a). *, P < 0.05; ***, P < 0.01.

FIG 7

Effect of JH-III on egg development and viability. (A) JH-III application reduces oviposition (*, P < 0.001). (B) Representative images showing increased number of ovarioles developing in ovaries dissected from females that were exposed to JH-III. (C) Number of hatched eggs (fertility) is significantly reduced upon JH-III treatment compared with control psyllid eggs. (D) Representative image showing a nymph hatching from Ca. L. solanacearum+ control egg and a dehydrated egg laid by JH-III treated females. White arrow shows the egg shell from which the nymph hatched.