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 propagates efficiently in its vector, the Asian citrus psyllid, Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut is fundamental to improve current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles by endocytosis, termed Liberibacter containing vacuoles (LCVs). Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). rLCVs then further develop into bigger 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 employs endo/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 controlling can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

Agrobacterium-mediated transformation (AMT) was compared with a particle bombardment (PB) for stable transformation of `Eden Gem', a melon with high embryogenic potential. Pretreated cotyledonary explants were either wounded via particle bombardment prior to Agrobacterium infection or were bombarded with plasmid-coated gold microparticles, using a modified particle inflow-type gun. Although similar numbers of embryos initially were obtained with each method, most produced via AMT became abnormal, possibly due to the growth-regulatory effects of the antibiotic mefoxine, which was used to inhibit Agrobacterium. Stably transformed plants and progeny were obtained only with PB, as determined by detection of the NPTII gene in R0 plant by Southern hybridization and, in progeny, by PCR amplification.

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Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.