Zhou, Changyong

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.

Citrus Huanglongbing (HLB), a devastating disease caused by the Gram-negative bacterium ‘ Candidatus Liberibacter asiaticus’ ( C Las), poses serious threats to global citrus production and lacks effective control strategies. Previously, SDE2470 (CLIBASIA_02470) was identified as a Sec-dependent effector that contributes to C Las pathogenesis, although its underlying molecular mechanisms were not fully elucidated. In this study, SDE2470 was found to target a citrus vascular one-zinc-finger transcription factor CsVOZ2. CsVOZ2 overexpression ( CsVOZ2- OE) in transgenic citrus plants significantly suppressed C Las colonization, whereas its RNA interference (RNAi) in citrus hairy roots enhanced susceptibility to C Las. Additionally, CsVOZ2 -OE significantly increased reactive oxygen species (ROS) and abscisic acid (ABA) contents accumulation and activated related genes expression. Further investigation revealed that the E3 ligase CsBTS1 directly interacts with CsVOZ2 and promotes its degradation via the 26S proteasome pathway. CsBTS1E3 -OE in citrus hairy roots markedly enhanced C Las proliferation. Importantly, SDE2470 directly interacts with CsBTS1E3 and strengthen CsBTS1E3-CsVOZ2 interaction. Meanwhile, SDE2470 strengthened the E3 ligase activity of CsBTS1, promoting CsVOZ2 degradation. Taken together, these findings support a model in which SDE2470 hijacks CsBTS1 to destabilize CsVOZ2, thereby disrupting ROS- and ABA-dependent immunity and promoting C Las infection in citrus.

ABSTRACT Citrus Huanglongbing (HLB), a devastating disease caused by the unculturable bacterium ‘ Candidatus Liberibacter asiaticus’ ( C Las), poses a severe threat to global citrus production. C Las secretes effectors to suppress host immune responses and facilitate its colonisation. Previously, the C Las effector SECP8 (CLIBASIA_05330) has been identified as an immune inhibitor. However, its molecular mechanisms on host immune suppression remain unclear. This study identifies the citrus transcription factor CsTCP15 as a target of SECP8. Transgenic citrus plants overexpressing CsTCP15 enhanced resistance to C Las, whereas CsTCP15 ‐RNAi interference plants became more susceptible, confirming its role as a positive immune regulator. Meanwhile, CsTCP15 was demonstrated to directly bind to cis ‐elements of salicylic acid (SA)‐responsive genes CsPR5 and CsWRKY22 , and overexpression of either gene strengthened citrus hairy roots' resistance against C Las. However, SECP8 directly interacts with CsTCP15 and inhibits its homodimerization. Concurrently, mSECP8 facilitates CsBRG3‐mediated degradation and further prevents the dimerization of CsTCP15. This two‐pronged interference eventually impairs the transcriptional activation of CsPR5 and CsWRKY22 , thereby compromising salicylic acid‐mediated immunity and promoting C Las infection. Our findings reveal a virulence strategy whereby a C Las effector manipulates a key host immune regulator to establish pathogenesis.

Abstract Citrus Huanglongbing (HLB) is the most destructive disease in citriculture, mainly caused by Candidatus Liberibacter asiaticus (CLas). However, the immune response of citrus to CLas at the cellular level remains to be elucidated. In this study, the first single-cell atlas of rough lemon (Citrus jambhiri Lush.) root apexes were generated using single-nucleus RNA sequencing at 20 weeks post-inoculation with CLas. According to gene expression patterns, the single-cell atlas was partitioned into 20 transcriptionally distinct clusters, and five cell types were identified within these clusters. A significant number of defense-related genes were co-upregulated across the five cell types following CLas infection, whereas genes involved in signal transduction pathways, such as tubulin beta-6 chain (TUBB1) and the phospholipase D alpha 1 (PLD1), were concurrently downregulated. Based on pseudotime trajectory analysis, the key pathways and genes involved in the coordination of cell differentiation and resistance in citrus under CLas infection were characterized. Following CLas infection, the development of phloem cells was significantly delayed, and the differentiation of cambium cells into xylem cells was evident. The expression of genes associated with lignin synthesis was significantly upregulated in these cells. The reduction in phloem cell differentiation and the enhanced differentiation of cambium cells into defense-related xylem cells may represent the primary vascular immune mechanisms exhibited by citrus plants in response to CLas infection. Additionally, DNA-binding one zinc finger transcription factor DOF2.4 was found to potentially serve dual roles in regulating vascular cell development and inducing plant resistance against CLas. In conclusion, this study collectively provides insights into the cellular innate immunity responses of citrus to CLas infection. These findings hold significant implications for the sustainable development of citriculture amidst the ongoing global HLB epidemic, and offer novel insights into vascular immunity and plant defense responses.

AbstractBACKGROUNDAutophagy is a conserved mechanism by which eukaryotic organisms defend against pathogen infection. However, the molecular mechanisms underlying the role of autophagy in the interactions of insect vectors with the phloem‐limited bacterial pathogen remain unclear. The citrus Huanglongbing (HLB)‐associated pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) seriously endangers development of the citrus industry. It spreads via Diaphorina citri in a persistent and propagative manner.RESULTSIn this study, a total of 30 autophagy‐related genes (ATG) were identified in the D. citri genome, among which multiple genes were significantly regulated after CLas infection. Concurrently, CLas infection also leads to an increased number of autophagosomes and enhanced accumulation of ATG8‐II. Ultrastructural observations revealed the presence of bacterial‐like structures within autophagosomes in the midgut of CLas‐infected D. citri. Furthermore, both activation and inhibition of autophagy significantly influenced CLas titers. However, autophagy cannot completely eliminate CLas in D. citri. We identified a CLas effector, SDE4040 (CLIBASIA_04040), that interacts with DcATG8 and co‐localizes on autophagosomes in D. citri. Co‐expression of SDE4040 and DcATG8 induces autophagy in Spodoptera frugiperda (Sf9) cells.CONCLUSIONTaken together, these results indicate that CLas infection activates the autophagy pathway in D. citri, contributing to a reduction in bacterial titer. Our data also revealed that CLas may trigger complex interactions with the insect. © 2025 Society of Chemical Industry.

Abstract Autophagy, a cellular process involved in the degradation and recycling of cellular components, has emerged as a pivotal mechanism for maintaining cellular homeostasis and combating pathogen invasion. Here, we provide evidence that the overexpression of CsATG8c inhibits CLas proliferation in citrus. CsATG8c directly interacts with the CLas effector protein SDE4040, leading to its degradation via the autophagic pathway. The SDE4040 protein acts as a virulence effector, and transgenic citrus plants expressing SDE4040 promote CLas proliferation by suppressing the hypersensitive response (HR) of the host and reducing the levels of jasmonic acid (JA). Collectively, these results demonstrate that ATG8-mediated selective autophagy limits CLas infection by degrading a virulence effector protein, which provides the first evidence for the role of autophagy in combating CLas infection.

Abstract Citrus Huanglongbing (HLB) is one of the most devastating diseases in the citrus industry and is caused primarily by ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-restricted gram-negative bacterium transmitted via citrus psyllids in the field. Precise CLas detection is crucial for HLB control, particularly during extensive surveys in new emerging regions. Unfortunately, the lack of a practical on-site detection method for CLas due to the limited specificity of immunostrips and the costive non-user friendliness approaches prevent the effective disease control. In this study, a probe-based recombinase polymerase amplification (RPA) targeting the CLas five-copy nrdB gene (β-subunit of ribonucleotide reductase, RNR) was developed and evaluated for its specificity, sensitivity, and reliability. To enhance user-friendliness and facilitate widely use, an All-in-one kit was premade as freeze-dried pellets in individual tubes, containing all necessary components except DNA template. The applicability of the All-in-one kit for CLas detection was confirmed using plant or psyllid crude DNA extracts. Collectively, the All-in-one kit offers a specific, sensitive, rapid, cost-effective, and practical alternative for diagnosing CLas in field conditions when coupled with simplified crude DNA extraction method, providing an alternative approach for on-site CLas detection in the context of HLB.