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.
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’ (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.
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.
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.
AbstractA major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.