HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation

Abstract

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as 'Candidatus Liberibacter asiaticus' that cause citrus greening disease (or Huanglongbing, HLB) and 'Ca. Liberibacter solanacearum' (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host' and vector's immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant's effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.

Keywords: Ca. Liberibacter solanacearum; HPE1; RAD23; effector; ubiquitin-proteasome system.

Figure 1

Sequence analysis of the HPE1 interacting clone, HIC1, identified in yeast two-hybrid screening. (A) Protein structure of the HIC1 and its full-length encoding protein, RAD23e. (B) Amino acid sequence alignment of the ubiquitin-like domain (UBL) of RAD23 proteins from Solanum lycoperisicum and Bactericera cockerelli. Asterisk, colon and dot denote the conserved and similar amino acid residues.

Figure 2

Validation of HPE1 and RAD23 interactions by directed Y2H. (A) Y2H test of interaction between SlRAD23e and the HPE1 from LsoA and LsoB. pGBKT7 (BD) constructs were used as bait, and pGADT7 (AD) constructs were used as prey in Y2H. Lam + SV40 indicates negative control for Y2H; p53 + SV40 indicates positive control for Y2H. (B) Interaction between HPE1 proteins and the other SlRAD23 proteins.

Figure 3

(A) HPE1B and RAD23e colocalized at the nuclei and cytosol of Nicotiana benthamiana epidermal cells. Upper panel: HPE1B:YFP:HA at 25 °C; Middle panel: HPE1B:YFP:HA at 32 °C; Lower panel: SlRAD23e:YFP:HA at 25 °C. Scale bar = 50 µm (B) Bi-molecule fluorescence complementation (BiFC) analysis of HPE1B and RAD23e interaction in N. benthamiana epidermal cells infiltrated with a 1:1 ratio of agrobacteria containing HPE1B-YC and RAD23e-YN. Images were taken between 48–60 h postinfiltration.

Figure 4

(A) BcRAD23e and HPE1B colocalize in the nucleus and cytosol of cultured Tni insect cells. (A) Transient expression of a red fluorescent chimera of BcRAD23 (RAD23:mCherry). (B) Transient expression of a green fluorescent chimera of HPE1B (HPE:EGFP). (C) Colocalization of the RAD23:mCherry and HPE:EGFP fluorescent signals in the cytosol and nucleus. Colocalized signals in discrete punctae were also observed in some cells (lower panel insets). Images were taken 48 h post-transfection with NucBlue used as a counterstain for the nucleus. Scale bar = 20 µm.

Figure 5

Immunoblot of heterologous expressing of HPE1 in N. benthamiana. (A) HPE1B is not ubiquitinated in the plant cell, but is degraded in a proteasome-dependent manner. MG132 + and MG132 —indicate treatment of the proteasome inhibitor, MG132. (B) Heterologous expression of HPE1B increased the accumulation of ubiquitinated proteins in N. benthamiana. HPE1B = HPE1B-YC, EV: empty vector. hpi indicates hours postinfiltration. (C) Coexpression of HPE1B and SlRAD23e further decreases the stability of HPE1 proteins in N. benthamiana. SlRAD23e = SlRAD23e-YN Arrow indicates the expected size of HPE1B fusion protein.

Figure 6

Immunoblot of tomato leaves at 4 weeks and 6 weeks post-LsoB infection. (A) Plants were infested with either Lso-free (Mock) or LsoB-infected (LsoB) adult psyllids. The dotted box indicates the range (60–200 kDa) used to quantify the ubiquitin signal intensity in (B). Error bars indicate the standard deviation (SD). Asterisk indicates significant difference from Mock group by Student’s t-test (p < 0.05). The experiment was replicated three times, each with three plants in each group. The Western blot was repeated at least twice per sample, each showing similar results.

Figure 7

Lso colonization in wild-type and HPE1 transgenic tomato plants. (A) PCR detection of Lso at 2-, 3-, and 4-week postinfection. WT, wild-type; FL, full-length HPE1B; MP, mature protein HPE1B. Arrowhead indicates the expected band of the Lso PCR product. (B) Symptom development was examined at 7 weeks postinfection. Arrowhead indicates the necrosis symptom of the shoot tip observed. Scale bar = 1 cm.