Wang, Chunxia

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most destructive citrus disease worldwide, severely reducing yield and fruit quality. Although no naturally resistant cultivars are available, citrus plants overexpressing Arabidopsis NPR1 ( At NPR1) display enhanced tolerance, yet the molecular mechanisms underlying this phenotype remain insufficiently understood. To uncover early transcriptional events associated with tolerance, we performed comparative RNA-seq and phytohormone profiling of susceptible wild-type (WT) and tolerant At NPR1-overexpressing ( At NPR1-OE) Duncan grapefruit at 0, 6, and 24 hours post infection (hpi). Before infection, At NPR1 plants downregulated genes involved in cytoskeleton organization, cell wall biogenesis, and receptor signaling, suggesting a primed basal defense state. After CLas exposure, At NPR1 plants exhibited stronger and earlier transcriptional reprogramming, with substantially more differentially expressed genes at 6 hpi than WT. At 24 hpi, At NPR1 plants showed suppression of callose synthase genes and selective induction of β-1,3-glucanases, indicating more controlled phloem callose regulation. Concurrently, attenuated expression of respiratory burst oxidase homologs and ROS-associated genes suggested a moderated and less damaging oxidative burst. At NPR1 plants maintained stable levels of salicylic acid, and gibberellins while preventing the CLas-induced induction of abscisic acid observed in WT. Collectively, these findings reveal that At NPR1 overexpression enhances HLB tolerance by integrating early transcriptional reprogramming with balanced structural, oxidative, and hormonal responses. This study provides a mechanistic framework for understanding NPR1-mediated tolerance to CLas during the initial stages of infection.

Abstract Huanglongbing (HLB), a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas), triggers persistent immune activation marked by excessive callose deposition and reactive oxygen species (ROS) accumulation, which impairs phloem function. This maladaptive response has led to HLB being described as a “pathogen-triggered immune disease”. Overexpression of the Arabidopsis (Arabidopsis thaliana) NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (AtNPR1) gene, a master regulator of systemic acquired resistance (SAR), confers robust HLB tolerance in susceptible citrus varieties, with transgenic lines exhibiting minimal or no disease symptoms following CLas infection. However, the mechanism underlying this tolerance remains unclear. In this study, we demonstrate that AtNPR1 restores immune homeostasis in CLas-infected trees by suppressing callose and ROS hyperaccumulation, thereby alleviating HLB symptom development. Similarly, silencing the Citrus sinensis homolog of NPR3 (CsNPR3), a negative regulator of SAR, mitigates CLas-induced immune overactivation and enhances HLB tolerance. Notably, salicylic acid (SA) levels are lower in AtNPR1-overexpressing citrus plants than wild-type controls after CLas infection, consistent with NPR1's role in negative feedback regulation of pathogen-induced SA accumulation. In Arabidopsis, overexpression of AtNPR1 or disruption of AtNPR3/AtNPR4 also attenuates pathogen-induced callose and ROS responses. Together, these findings reveal conserved roles for NPR1/NPR3/NPR4 in immune regulation across species and suggest that HLB susceptibility in commercial citrus varieties stems from a diminished capacity to maintain immune balance.

‘ 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. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

ABSTRACT‘CandidatusLiberibacter 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 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 to 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.FundingThis work is supported by USDA National Institute of Food and Agriculture award No. 2020-70029-33197 to W.M and A.L.

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

AbstractHuanglongbing (HLB) causes significant economic loss in citrus production worldwide. HLB is caused by Candidatus Liberibacter asiaticus (CLas), a gram-negative bacterium which inhabits the phloem exclusively. CLas infection results in accumulation of callose and reactive oxygen species in the phloem of infected plants, but little is known about the specific processes that take place during infection because of the sparse distribution of bacteria and the inaccessibility of the phloem inside the tree. In this study, we used the seed vasculatures, which accumulate a high number of CLas, as a model tissue to study CLas-host cellular interactions. In vasculature where CLas is abundant, sieve pore callose and H2O2 concentration were reduced compared to healthy seed vasculature. The expression of callose synthases (CalS) and respiratory burst oxidase homolog (RBOH) genes were downregulated in infected seeds compared to healthy ones. In leaves of HLB-infected plants, H2O2 concentration and CalS expression increased compared to uninfected leaves, but cells with CLas had lower levels of sieve plate callose compared to cells without CLas. Our results provide evidence that the bacteria manipulate cell metabolism to disable plant defenses and suggests that HLB disease is the result of a constant arms-race between the pathogen and a defense response, which is ultimately harmful to the host plant.

‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

AbstractIn Florida, almost all citrus trees are infected with Huanglongbing (HLB), caused by the gram-negative, intracellular phloem limited bacteria Candidatus liberibacter asiaticus (CLas). Distinguishing between the severely and mildly sick trees is important for managing the groves and testing new HLB therapies. A mildly sick tree is one that produces higher fruit yield, compared to a severely sick tree, but measuring yields is laborious and time consuming. Here we characterized HLB affected sweet orange trees in the field in order to identify the specific traits that are correlated with the yields. We found that canopy volume, fruit detachment force (FDF) and the percentage of photosynthetically active radiation interception in the canopy (%INT) were positively correlated with fruit yields. Specifically, %INT measurements accurately distinguished between mild and severe trees in independent field trials. We could not find a difference in the Ct value between high and low producing HLB trees. Moreover, Ct values did not always agree with the number of CLas in the phloem that were visualized by transmission electron microscopy. Overall, our work identified an efficient way to distinguish between severe and mild HLB trees in Florida by measuring %INT and suggests that health of the canopy is more important for yields than the Ct value.

AbstractCandidatus Liberibacter asiaticus (Las) is an emergent bacterial pathogen that is associated with the devastating citrus Huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful and dual-transcriptome analyses aiming to profile gene expression in both Las and its host(s) have a low coverage of the Las genome due to the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, the lack of a Las transcriptome remains as a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited the highest expression levels in citrus include ion transporters, ferritin, outer membrane porins, and genes involved in phage-related functions, pilus formation, cell wall modification, and stress responses. One hundred and six genes were found to be differentially expressed in citrus vs psyllids. Genes related to transcription/translation and resilience to host defense response were upregulated in citrus; whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. We also determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.