The portal includes user-friendly manual curation tools to allow the research community to continuously improve the knowledgebase as more experimental research is published. Bulk downloads are available for all genome and annotation datasets from the FTP site.The inability to culture the huanglongbing -associated pathogen Candidatus Liberibacter asiaticus , has provided significant challenges for understanding the molecular mechanisms controlling disease progression. To provide insight on how CLas manipulates citrus during infection, we used a comparative proteomics approach to identify dynamic changes in the phloem proteome of navel sweet oranges. Navels were mock or graft inoculated with the California CLas strain HHCA. At ten months post inoculation, crude phloem was extracted from uninfected and infected navel trees. Differentially expressed proteins were identified using label-free quantitative mass spectrometry. We identified various protease classes such as glycosidases, serine proteases, cysteine proteases, and aspartic proteases as significantly induced. Homologous proteins act as immune-related proteases in Arabidopsis, tomato, and maize and are targeted by diverse pathogens to promote infection. The over expression or deletion of specific proteases in crops such as maize, tomato and potato have shown to have enhanced resistance and susceptibility to pathogens, respectively. To identify whether the identified proteases are also induced in citrus field samples, we acquired uninfected and infected samples from a citrus grove in Texas. We used mass spectrometry to quantify protease abundance. Additionally,hydroponic grow system we assayed for activity using activity-based protein profiling . ABPP permits the quantification and identification of active proteases.

Our current data demonstrates that the abundance and activity of several proteases is increased in infected samples. A specific group of proteases display enhanced abundance but no altered activity during infection. Based on these observations, we hypothesize that CLas suppresses citrus defense responses by targeting host protease activity. Identifying the role of citrus proteases will contribute to the development of HLB biomarkers and specific proteases could be manipulated to enhance resistance to CLas. Huanglongbing is a widespread and devastating citrus disease. The HLB-associated pathogen, Candidatus Liberibacter asiaticus is transmitted by the Asian citrus psyllid, a piercing-sucking insect. The Asian citrus psyllid feeds on citrus phloem and can acquire CLas by feeding on an infected tree. In this study, we used mass spectrometry and quantitative proteomics to identify proteins that were differentially expressed in navel phloem during infection. Multiple differentially expressed proteins were proteases and we were able to identify the same classes of proteases as differentially expressed in infected navel trees sampled from the field in Texas. These proteins have the potential to serve as biomarkers for HLB as well as targets that can be manipulated for disease control. We have also investigated the genetic diversity and conservation of predicted secreted proteins present in CLas. We analyzed eight currently available genome sequences and sequenced two new strains from China and Florida. Using stringent criteria, we identified a group of CLas secreted proteins that are conserved across all sequenced strains. Collectively, the completion of this research has enabled a greater understanding of how the plant responds to CLas infection as well as identified plant and bacterial components with promise for pathogen detection and control. Up to now, cultivation of citrus greening pathogens has been attempted in many laboratories. “Candidatus Liberibacter asiaticus ” is known as a fastidious bacterium that is unculturable in many traditional culture media. To facilitate basic research and establish definitive diagnosis, we are developing a new selective culture medium for Las and the other related species.

At first, we examined whether Las can multiply in some traditional media and Las specific media reported previously. Living Las cells were extracted from the infected citrus leaves according to our procedure which was developed in our previous reports. The multiplication of Las cells was evaluated from the copy number of Las DNA amplified using real-time PCR. The results indicated that almost existing media do not support the multiplication of Las cells enough. Thus we explored constitutions of a new Las selective medium. By comparing with other bacterial genomes, inability of Las genome to produce essential metabolic pathway was predicted. Then based on this prediction, we have developed a culture medium by adding the compounds to replenish the shortage of nutrients for Las. And a new medium could induce the multiplication of Las cells. But multiplication was very slow and there was no visible typical colony on a solid medium. In spite of lack of visible colonies, after 14 days of incubation, Las cells were detected by in situ hybridization. Moreover, RNA-seq, RT-PCR and partially DNA sequencing could be performed from the invisible agents on the medium. Towards establishing the practical and reproducible method of cultivation, we continue to improve the medium. Response of Candidatus Liberibacter asiaticus to antibiotic treatment in vitro was demonstrated experimentally using a new medium. Primarily, a metabolic pathway analysis of Ca. L. asiaticus genome was carried out using KEGG database. Comparative approaches between Ca. L. asiaticus and closely-related bacterial species allowed us to identify a group of genes that do not remain in the pathogen’s genome and select essential nutrimental components for the preparation of a new culture medium, including sugar, amino acids, fatty acids and vitamins that cannot be synthesized by the pathogen. Quantification of Ca. L. asiaticus was investigated for a month using real time PCR assays and in situ hybridization. Ca. L. asiaticus became detectable two weeks after initiating incubation and thereafter. Very small colony-like patterns appeared in the media, but the further growth was not observed. Furthermore, in-vitro assays to examine pathogen’s responses to eight antibiotics was conducted using the new medium. The results showed that Ca. L. asiaticus did not proliferate in the culture medium supplemented with polymyxin B. On the other hand, supplement of tetracycline or oxytetracycline in the medium resulted in accelerating the growth of the pathogen.

The mode of action of tetracycline and oxytetracycline on Ca. L. asiaticus remained unknown. In short, this study suggests that a culture-dependent approach may give us vital insights into further understanding of the behavior of Ca. L. asiaticus and contribute to the screening of potential agents and chemicals for citrus greening disease management. Development of methods to fight Huanglongbing , caused by “Candidatus Liberibacter asiaticus” , has been hampered by lack of ability to culture CLas in the lab, which precludes the testing of gene functions that may be involved in CLas viability or virulence. Identification of such gene functions will have an enormous impact on the ability to control CLas and eventually allow development of integrated strategies to stop HLB spread by the Asian citrus psyllid and the resulting economic devastation to citrus agriculture. Development of the ability to culture CLas in vitro will open up new avenues of research that are guaranteed to provide a game change in knowledge of CLas biology and transmission, including a culturing platform necessary for development of an amenable genetic system. The Ca. Liberibacter Culturing Consortium was established to develop such an in vitro culture system and make it available to the citrus industry and academic research community by establishing a system for host cell-free culture of CLas, providing standard operating procedures for culture of CLas to the research community, and providing cultures through standard repositories. Progress to date in these efforts will be reported.Candidatus Liberibacter asiaticus , the most prevalent causal agent of huanglongbing citrus disease in North America and Asia, is a phloemlimited and unculturable bacterium. However, during the past few years,ebb flow table genomes of several Liberibacter species were sequenced suggesting that members of this genus have suffered a significantly genome reduction. Comparative genomics of Las has allowed the prediction of the putative effector proteins, potentially involved in virulence. In this scenery, where small secreted protein could play crucial roles between plant and pathogen interaction, effector proteins identification is a complex objective. Plant defense can be triggered by the host perception of conserved pathogen-associated molecular patterns . Pathogens in turn can suppress PAMP-triggered immunity, causing disease. Identification of Las effector proteins is a key step to understand the mechanisms by which Las disrupt the host response. To provide a functional basis for this hypothesis, eight of these sequences were cloned and expressed fused to green or red fluorescent proteins under the control of 35S promoter. Agrobacterium-mediated transient expressions were performed in Nicotiana tabacum and N. benthamiana leaves. Bacterial protein expressions were monitored over a 10-day period by fluorescence and confocal laser scanning microscopy. All tested genes shown expression in plant cells, and revealed different subcellular distribution patterns respect to the control. CLIBASIA_04560 encoded a hypothetical protein which is localized in nucleus when it is transiently expressed in N. benthamiana. More interesting, this protein triggers an increase of H2O2 production respect to the control without necrotic or cell death phenotype in N. benthamiana after 3 dpi.

Now, we are studying if Clibasia_04560 may act as an effector protein modulated the energetic metabolism in citrus plants. Asian citrus psyllid is a known vector of Huanglongbing disease that is an imminent threat to the California citrus industry. At present, controlling the psyllid vector is the best management strategy to delay the spread of HLB, while disease management tools are developed. In recent years ACP finds from trap catches have increased in the San Joaquin Valley especially in residential areas and at packinghouses and juice plants. Researchers have attributed HLB spread in Florida to human-assisted transport of infected psyllids and infected plant material including bulk citrus. ACP quarantine boundaries and HLB quarantine boundaries have been established in California and preharvest treatments are required to ship insect-free bulk citrus between quarantined zones to mitigate the spread of ACP and eventually HLB. It is clear from the finds at juice plants and packinghouses that current methods of orchard disinfestation are not reducing psyllids sufficiently and psyllids are being moved in bulk citrus In the context of identifying a postharvest treatment to control ACP after harvest and before shipping to packinghouses, University of California Researchers in collaboration with USDA-ARS and Fruit Growers and Supply have begun testing the potential of fumigation on Asian Citrus Psyllid to develop a treatment plan for disinfesting bulk citrus. We discuss the prospects for use of postharvest fumigation to disinfest bulk citrus prior to shipping to other regions of the state. Asian citrus psyllid feeding behaviors play a significant role in the transmission of the phloem-limited Candidatus Liberibacter asiaticus bacterium that causes the economically devastating citrus greening disease. Recent studies have shown a fibrous ring of thick-walled sclerenchyma around the phloem in mature citrus leaves that is more prominent on the lower compared with the upper side. We performed Electrical Penetration Graph studies on ACP adults placed on lower or upper surfaces of young or mature Valencia orange leaves. Feeding sites on the same leaf tissues were then sectioned and examined by epifluorescence microscopy. Based on the EPG recordings and histological correlations, we found that thick-walled fibrous sclerenchyma located around the phloem of mature citrus leaves significantly reduced phloem ingestion by psyllids placed on the lower leaf surface compared with ingestion from the upper surface of mature leaves or on young leaves. The longest duration of phloem ingestion was observed from the upper side of young flush leaves that had the least developed sclerenchyma. Bouts of phloem salivation , however, were significantly longer on mature leaves compared with young flush. ACP adults made consecutive phloem feeding attempts on the lower side of mature leaves and those bouts resulted in unsuccessful or shorter periods of phloem ingestion. ACP adults also made more frequent and longer bouts of xylem ingestion on mature leaves compared with psyllids placed on young leaves. Our results support the hypothesis that the presence of a thick, well-developed fibrous ring around phloem tissues of mature leaves acts as a barrier to frequent or prolonged phloem ingestion by ACP from citrus leaves. This may have an important role in limiting or preventing CLas acquisition and/or transmission by ACP, and could be used for identification and development of resistant citrus cultivars. Phytophagous insects including Asian citrus psyllids use multiple sensory modalities [vision, olfaction, contact chemoreception, gustation , perception of auditory or vibrational stimuli] to locate host plants or conspecifics.