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.

CLIBASIA_00460 Disrupts Hypersensitive Response and Interacts with Citrus Rad23 Proteins

“…Overall, these studies show that the localization of CLIBASIA_00460 can be affected by abiotic conditions, and this should be taken into consideration when evaluating the development of HLB under different conditions. Previously, the effect of temperature on subcellular distribution was not observed for Lso-HPE1 [ 28 ]. Lso-HPE1 and CLIBASIA_00460 mature proteins share 60% amino acid sequence similarity; these differences could be linked to the differences in protein localization associated with temperature or the interaction with RAD23 proteins.…”

“…These results were confirmed using BiFC. An analysis of Lso HPE1 and S. lycopersicum RAD23 proteins also identified the interaction between the effector protein and the RAD23 proteins in Clade 2, but not in Clade 1 [ 28 ]. A mutant analysis determined that in A. thaliana , at least one functional RAD23 gene is required for plant viability, independent of the RAD23 subfamily [ 31 ].…”

“…Thus, the subcellular localization pattern was changed when CLIBASIA_00460 interacted with C. sinensis RAD23c or RAD23d proteins. Lso-HPE1 also interacted with S. lycopersicum RAD23 proteins, but there was no change in the localization of the proteins; the interaction occurred in the nucleus and cytoplasm [ 28 ]. This difference in the localization of CLIBASIA_00460 when interacting with the C. sinensis RAD23 proteins was unexpected because the mature Lso-HPE1 and CLIBASIA_00460 proteins, as well as the different RAD23 proteins, were predicted to be localized in the nucleus and the cytoplasm.…”

“…Lso-HPE1 is a Sec-dependent secreted protein that is able to suppress a hypersensitive response (HR) when induced by either BAX or Prf D1416V in Nicotiana benthamiana [ 27 ]. The screening of a tomato yeast two-hybrid library identified tomato RAD23e as an interactor of Lso-HPE1 [ 28 ]. RAD23 proteins are involved in shuttling ubiquitinated proteins to the proteasome for them to undergo degradation.…”

Comparative genomics of the Liberibacter genus reveals widespread diversity in genomic content and positive selection history

“…Moreover, through Blast analyses we recovered annotation evidence for two other genes: group_2579 was annotated as a metallophosphoesterase and group_2831 as hypothetical protein effector 1 (HPE1). HPE1 has been experimentally shown to suppress plant effector-triggered immunity in Lso infection ( Kan et al, 2021 ). Our results corroborate the potential importance of this effector family based on a history of positive selection.…”