Candidatus Liberibacter solanacerum (CLso), transmitted by Bactericera trigonica in a persistent and propagative mode causes carrot yellows disease, inflicting hefty economic losses. Understanding the process of transmission of CLso by psyllids is fundamental to devise sustainable management strategies. Persistent transmission involves critical steps of adhesion, cell invasion, and replication before passage through the midgut barrier. This study uses a transcriptomic approach for the identification of differentially expressed genes with CLso infection in the midguts, adults, and nymphs of B. trigonica and their putative involvement in CLso transmission. Several genes related to focal adhesion and cellular invasion were upregulated after CLso infection. Interestingly, genes involved with proper functionality of the endoplasmic reticulum (ER) were upregulated in CLso infected samples. Notably, genes from the endoplasmic reticulum associated degradation (ERAD) and the unfolded protein response (UPR) pathway were overexpressed after CLso infection. Marker genes of the ERAD and UPR pathways were also upregulated in Diaphorina citri when infected with Candidatus Liberibacter asiaticus (CLas). Upregulation of the ERAD and UPR pathways indicate induction of ER stress by CLso/CLas in their psyllid vector. The role of ER in bacteria–host interactions is well-documented; however, the ER role following pathogenesis of CLso/CLas is unknown and requires further functional validation.

CLas inhibits callose deposition in the sieve pores and the accumulation of reactive oxygen species to favor its cell-to-cell movement.

Transient gene expression is a powerful tool to study gene function in plants. In citrus, Agrobacterium transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to Agrobacterium transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than Agrobacterium. Here we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of Xanthomonas citri effector AvrGF1. To further expand the available tools for subcellular localization studies in citrus, we also generated a new set of transgenic citrus plants that contain organelle markers labelling the nuclei, actin and endoplasmic reticulum. Using these new tools, we were able to show that the coat protein of citrus tristeza virus localizes to the cytoplasm and nuclei when expressed in epidermal cells fused to GFP. We have optimized a new method for transient expression in citrus leaves, to give highly reproducible and efficient transformation without producing a high level of injury or artifacts to the bombarded tissue. We also generated the first set organelle markers for use in citrus. These fluorescent protein markers label the nucleus and the actin. With these new resources, protein activity and subcellular localization can be studied in citrus rapidly and in high throughput. The handheld gene-gun device can also be used in the grove to deliver therapies for citrus diseases, such as canker and Huanglongbing, into trees.

In Florida, almost all citrus trees are affected with Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas). We characterized various parameters of HLB-affected sweet orange trees in response to yield-improving nutritional treatment, including canopy volume, canopy density and CLas Ct values, and found that the treatment improved yield and maintained canopy density for over three years, whereas untreated HLB-affected trees declined in canopy density. The nutritional treatment did not affect CLas titer or the tree canopy volume suggesting that canopy density is a better indicator of fruit yield. To further validate the importance of canopy density, we evaluated three independent orchards (different in tree age or variety) to identify the specific traits that are correlated with fruit yields. We found that canopy density and fruit detachment force (FDF), were positively correlated with fruit yields in independent trials. Canopy density accurately distinguished between mild and severe trees in three field trials. High and low producing HLB trees had the same Ct values. Ct values did not always agree with CLas number in the phloem, as visualized by transmission electron microscopy. Our work identifies canopy density as an efficient trait to predict yields of HLB-affected trees and suggests canopy health is more relevant for yields than the CLas population.

‘ Candidatus Liberibacter asiaticus’ (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to the citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen's biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared with previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib, and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .