Wang, Nian

Citrus huanglongbing (HLB), which is caused by the phloem-colonizing bacteria Candidatus Liberibacter asiaticus (CLas), poses a significant threat to citrus production worldwide. The pathogenicity mechanism of HLB remains poorly understood. SEC-dependent effectors (SDEs) have been suggested to play critical roles in the interaction between citrus and CLas. Here, we explored the function of CLIBASIA_05320 (SDE19), a core SDE from CLas, and its interaction with its host target. Our data revealed that SDE19 is expressed at higher level during infection of citrus than that during infection of the Asian citrus psyllid. Subcellular localization assays showed that SDE19 is localized in the nucleus and cytoplasm and is capable of moving from cell to cell in Nicotiana benthamiana. To investigate whether SDE19 facilitates pathogen infection, we generated transgenic Arabidopsis thaliana and citrus plants overexpressing SDE19. Transgenic A. thaliana and citrus plants were more susceptible to Pseudomonas syringae pv. tomato (Pst) and Xanthomonas citri subsp. citri (Xcc), respectively. In addition, RNA-seq analysis demonstrated that overexpression of SDE19 resulted in a reprogramming of expression of genes related to biotic stimulus responses. SDE19 interacts with Citrus sinensis Sec12, a guanine nucleotide exchange factor responsible for the assembly of plant COPII (coat protein II)-coated vesicles, which mediate vesicle trafficking from the ER to the Golgi. SDE19 colocalizes with Sec12 in the ER by binding to its N-terminal catalytic region, affecting the stability of Sec12 through the 26S proteasome. This interaction hinders the secretion of apoplastic defense-related proteins such as PR1, P69B, GmGIP1, and RCR3. Furthermore, the secretion of PR1 and callose deposition is decreased in SDE19-transgenic A. thaliana. Taken together, SDE19 is a novel virulent SDE secreted by CLas that interacts with Sec12 to disrupt vesicle trafficking, inhibit defense-related proteins secretion, and promote bacterial infection. This study sheds light on how CLas manipulates the host vesicle trafficking pathway to suppress the secretion of defense-related proteins and interfere with plant immunity.

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

AbstractThe bacterium Candidatus Liberibacter asiaticus (CLas) causes citrus Huanglongbing disease. Our understanding of the pathogenicity and biology of this microorganism remains limited because CLas has not yet been cultivated in artificial media. Its genome is relatively small and encodes approximately 1136 proteins, of which 415 have unknown functions. Here, we use a high-throughput yeast-two-hybrid (Y2H) screen to identify interactions between CLas proteins, thus providing insights into their potential functions. We identify 4245 interactions between 542 proteins, after screening 916 bait and 936 prey proteins. The false positive rate of the Y2H assay is estimated to be 2.9%. Pull-down assays for nine protein-protein interactions (PPIs) likely involved in flagellar function support the robustness of the Y2H results. The average number of PPIs per node in the CLas interactome is 15.6, which is higher than the numbers previously reported for interactomes of free-living bacteria, suggesting that CLas genome reduction has been accompanied by increased protein multi-functionality. We propose potential functions for 171 uncharacterized proteins, based on the PPI results, guilt-by-association analyses, and comparison with data from other bacterial species. We identify 40 hub-node proteins, including quinone oxidoreductase and LysR, which are known to protect other bacteria against oxidative stress and might be important for CLas survival in the phloem. We expect our PPI database to facilitate research on CLas biology and pathogenicity mechanisms.

AbstractCandidatusLiberibacter asiaticus (CLas) is the causal agent of the devastating citrus Huanglongbing (HLB) disease. Our understanding of the pathogenicity mechanism and biology of CLas remain limited because CLas has not been cultured in artificial media. CLas encodes 1136 proteins of which 415 have unknown functions. Since genetic studies of CLas genes with unknown functions are impossible, we utilized genome-wide protein-protein interactions (PPIs) yeast-two-hybrid (Y2H) assays to help solve the mystery. PPIs are fundamental to all cellular processes and machinery and instrumental in investigating uncharacterized proteins and inferring biological pathways. In total, 916 bait and 936 prey proteins were included in the three-phase screening, which identified 4245 interactions. The false positive rate of the Y2H assay was estimated to be 3.1%. Pull-down assays confirmed the robustness of our Y2H. The average interactions per node for CLas Y2H interactome were approximately 15.6, significantly higher than free-living bacteria, indicating genome reduction has led to a multi-function of proteins. PPIs provide clues for functions of 371 uncharacterized proteins of CLas. Forty HUB node proteins were identified which might play critical roles in CLas, including a quinone oxidoreductase and LysR that are known to protect bacteria against oxidative stress. This explains why CLas survives well in the phloem even though it triggers immune-mediated disease, systemic and chronic production of reactive oxygen species, and phloem cell death. This PPI database facilitates the investigation of CLas cellular biochemistry and physiology, functions of uncharacterized proteins, and pathogenicity mechanisms of the pathogen.

‘ Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant−pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant−pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.

Citrus Huanglongbing (HLB) is the most devastating citrus disease in the world. Candidatus Liberibacter asiaticus (Las) is the prevalent HLB pathogen, which is yet to be cultivated. A recent study demonstrates that Las does not contain pathogenicity factors that are directly responsible for HLB symptoms. Instead, Las triggers systemic and chronic immune responses, representing a pathogen-triggered immune disease. Importantly, overproduction of reactive oxygen species (ROS) causes systemic cell death of phloem tissues, thus causing HLB symptoms. Because Las resides in the phloem tissues, it is expected that phloem cell might recognize outer membrane proteins, outer membrane vesicle (OMV) proteins and extracellular proteins of Las to contribute to the immune responses. Because Las has not been cultivated, we used Liberibacter crescens (Lcr) as a surrogate to identify proteins in the OM fraction, OMV proteins and extracellular proteins by liquid chromatography with tandem mass spectrometry (LC–MS/MS). We observed OMVs of Lcr under scanning electron microscope, representing the first experimental evidence that Liberibacter can deliver proteins to the extracellular compartment. In addition, we also further analyzed LC–MS/MS data using bioinformatic tools. Our study provides valuable information regarding the biology of Ca. Liberibacter species and identifies many putative proteins that may interact with host proteins in the phloem tissues.

Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca. Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca. Liberibacter spp. infection and how Ca. Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca. Liberibacter spp. imply the involvement of immune response in the Ca. Liberibacter pathosystems. Lastly, we reviewed how Ca. Liberibacter spp. movement inside and interactions with plants lead to symptom development.