Candidatus Liberibacter asiaticus (CLas) causes 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 (TEM) analysis showed 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 the following categories: plant-pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling that are commonly associated with pathogen infections compared to 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. Additionally, qRT-PCR based expression analysis temporally visualized the induced expression of companion cell specific genes, phloem protein 2 (PP2) genes, and sucrose transport genes in young flush 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.
Citrus Huanglongbing (HLB), also known as citrus greening, is one of the most devastating citrus diseases worldwide. Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain associated with HLB, which is yet to be cultured in vitro. None of the commercial citrus cultivars are resistant to HLB. The pathosystem of Ca. Liberibacter is complex and remains a mystery. In this review, we focus on the recent progress in genomic research on the pathogen, the interaction of host and CLas, and the influence of CLas infection on the transcripts, proteins, and metabolism of the host. We have also focused on the identification of candidate genes for CLas pathogenicity or the improvements of HLB tolerance in citrus. In the end, we propose potentially promising areas for mechanistic studies of CLas pathogenicity, defense regulators, and genetic improvement for HLB tolerance/resistance in the future.
AbstractThe immune system is critical for keeping animals and plants healthy from pathogens. However, immune-mediated diseases are also common for human. Immune-mediated diseases have not been reported for plants. Here, we present evidence that citrus Huanglongbing (HLB), caused by phloem-colonizing Candidatus Liberibacter asiaticus (CLas), is an immune-mediated disease. CLas infection of Citrus sinensis stimulated systemic and chronic immune response in the phloem tissues including reactive oxygen species (ROS) production as indicated by H2O2, callose deposition, and induction of immune related genes. Systemic cell death of companion and sieve element cells, but not surrounding parenchyma cells, was observed following ROS production triggered by CLas. ROS production triggered by CLas localized in phloem tissues. The H2O2 concentration in exudates extracted from phloem enriched bark tissue of CLas infected plants reached a threshold of killing citrus protoplast cells, which was suppressed by uric acid (a ROS scavenger) and gibberellin. Foliar spray of HLB positive citrus with antioxidants (uric acid and rutin) and gibberellin significantly reduced both H2O2 concentrations and cell death in phloem tissues induced by CLas and reduced HLB symptoms. RNA-seq analyses of CLas infected and health C. sinensis support that CLas causes oxidative stress. In sum, HLB is an immune-mediated disease and both mitigating ROS via antioxidants and promoting plant growth can reduce cell death of the phloem tissues caused by CLas, thus controlling HLB.
‘Candidatus Liberibacter asiaticus’ (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old ‘Valencia’ sweet orange on ‘Kuharske’ citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 109 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 1010 cells/tree, and 6.0 × 1010 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 106 cells/leaf or 9.2 × 104 cells cm–2 in leaves, approximately 104 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.
Streptomycin (STR) has been used to control citrus huanglongbing (HLB) caused by ‘Candidatus Liberibacter asiaticus’ (CLas) via foliar spray. Here, we studied the residue dynamics of STR and its effect on CLas titers in planta applied by foliar spray and trunk injection of 3-year-old citrus trees that were naturally infected by CLas in the field. After foliar spray, STR levels in leaves peaked at 2 to 7 days postapplication (dpa) and gradually declined thereafter. The STR spray did not significantly affect CLas titers in leaves of treated plants as determined by quantitative PCR. After trunk injection, peak levels of STR were observed 7 to 14 dpa in the leaf and root tissues, and near-peak levels were sustained for another 14 days before significantly declining. At 12 months after injection, moderate to low or undetectable levels of STR were observed in the leaf, root, and fruit, depending on the doses of STR injected, with a residue level of 0.28 µg/g in harvested fruit at the highest injection concentration of 2.0 µg/tree. CLas titers in leaves were significantly reduced by trunk injection of STR at 1.0 or 2.0 g/tree, starting from 7 dpa and throughout the experimental period. The reduction of CLas titers was positively correlated with STR residue levels in leaves. The in planta minimum effective concentration of STR needed to suppress the CLas titer to an undetectable level (cycle threshold ≥36.0) was 1.92 µg/g fresh weight. Determination of the in planta minimum effective concentration of STR against CLas and its spatiotemporal residue levels in planta provides the guidance to use STR for HLB management.
‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus huanglongbing (HLB), colonizes inside the phloem and is naturally transmitted by the Asian citrus psyllid (ACP). Here, we investigated spatiotemporal CLas colonization in different tissues after ACP transmission. Of the nine plants successfully infected via ACP transmission, CLas was detected in the roots of all trees at 75 days postremoval of ACPs (DPR) but in the mature leaf of only one tree; this finding is consistent with the model that CLas moves passively from source to sink tissues. At 75 and 365 DPR, CLas was detected in 11.1 and 43.1% of mature leaves not fed on by ACPs during transmission, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates that CLas is capable of both passive and active movement, with passive movement being dominant. At 225 DPR, leaves fed on by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and the highest CLas titer, followed by leaves that emerged after ACP removal and mature leaves not fed on by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes 3 years at most for CLas to infect the whole tree. Overall, spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development and provides evidence showing that young leaves should be the focus of HLB management.