# Molecular Characterization of Endoplasmic Reticulum (ER) Stress-Associated BiP, IRE1, and XBP1 Genes in Diaphorina citri and Their Roles During Candidatus Liberibacter asiaticus Infection

## TL;DR

This study explores how ER stress genes in the citrus psyllid insect respond to a bacterial pathogen, revealing new insights into host-pathogen interactions.

## Contribution

The study identifies conserved IRE1-mediated XBP1 splicing as a reliable ER stress marker in Diaphorina citri and reveals its role in Candidatus Liberibacter asiaticus infection.

## Key findings

- ER stress in Diaphorina citri is marked by IRE1-mediated XBP1 splicing, a conserved UPR indicator.
- CLas infection triggers early UPR activation followed by suppression, with ER stress induction increasing bacterial titers.
- IRE1 and XBP1 have opposing effects on CLas proliferation, as revealed by RNAi silencing experiments.

## Abstract

Although endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR) are well characterized in mammalian systems, their role in plant pathogens transmitted by insect vectors remains poorly understood. In insects, ER stress genes are not well characterized, leading to a shortage of reliable markers or convenient methods to assess ER stress. Furthermore, the interaction between the citrus huanglongbing pathogen, Candidatus Liberibacter asiaticus (CLas), and its insect vector, Diaphorina citri, is unclear. In this study, we characterized three core UPR genes in D. citri and established conserved IRE1-mediated splicing of XBP1 mRNA as a reliable indicator of ER stress. During CLas infection, the UPR was dynamically regulated, showing early activation followed by later suppression. Functional assays demonstrated that ER stress induction increased CLas titers, while RNAi silencing revealed that IRE1 and XBP1 have different effects on bacterial proliferation. These findings elucidate a critical host–pathogen interaction and identify potential targets for disease management.

The endoplasmic reticulum (ER) stress response, or unfolded protein response (UPR), is crucial for cellular homeostasis and host defense. Its role in insect vectors of plant pathogens remains poorly understood. This study conducted a comprehensive molecular characterization of three core UPR genes—BiP, IRE1, and XBP1—in Diaphorina citri, the insect vector of the citrus huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas). Expression profiling showed distinct developmental and tissue-specific patterns for these genes. The IRE1-mediated unconventional splicing of XBP1 mRNA was identified in D. citri and predicted across diverse insect pests. A semi-quantitative RT-PCR assay was established to detect this splicing event for monitoring ER stress. Time-course analysis of CLas-infected D. citri revealed an early activation (upregulation of BiP, IRE1, XBP1 transcripts, and increased XBP1 splicing) followed by later suppression of the UPR. Functional studies demonstrated that induction of ER stress with thapsigargin increased CLas titer. RNAi-mediated silencing showed that IRE1 silencing increased CLas proliferation, whereas XBP1 silencing reduced it. These findings provide foundational insights into the ER stress pathway in D. citri and demonstrate that the IRE1-XBP1 branch of the UPR plays a critical role in modulating CLas infection dynamics within its insect vector.