Plant Science

In recent decades, ‘Candidatus Liberibacter spp.’ have emerged as a versatile group of psyllid-vectored plant pathogens and endophytes capable of infecting a wide range of economically important plant hosts. The most notable example is ‘Candidatus Liberibacter asiaticus’ (CLas) associated with Huanglongbing (HLB) in several major citrus-producing areas of the world. CLas is a phloem-limited α-proteobacterium that is primarily vectored and transmitted among citrus species by the Asian citrus psyllid (ACP) Diaphorina citri. HLB was first detected in North America in Florida (USA) in 2005, following introduction of the ACP to the State in 1998. HLB rapidly spread to all citrus growing regions of Florida within three years, with severe economic consequences to growers and considerable expense to taxpayers of the state and nation. Inability to establish CLas in culture (except transiently) remains a significant scientific challenge toward effective HLB management. Lack of axenic cultures has restricted functional genomic analyses, transfer of CLas to either insect or plant hosts for fulfillment of Koch’s postulates, characterization of host-pathogen interactions and effective screening of antibacterial compounds. In the last decade, substantial progress has been made toward CLas culturing: (i) three reports of transient CLas cultures were published, (ii) a new species of Liberibacter was identified and axenically cultured from diseased mountain papaya (Liberibacter crescens strain BT-1), (iii) psyllid hemolymph and citrus phloem sap were biochemically characterized, (iv) CLas phages were identified and lytic genes possibly affecting CLas growth were described, and (v) genomic sequences of 15 CLas strains were made available. In addition, development of L. crescens as a surrogate host for functional analyses of CLas genes, has provided valuable insights into CLas pathogenesis and its physiological dependence on the host cell. In this review we summarize the conclusions from these important studies.

‘Candidatus Liberibacter solanacearum’ is an unculturable α-proteobacterium that is the causal agent of zebra chip disease of potato—a major problem in potato-growing areas, because it affects growth and yield. Developing effective treatments for ‘Ca. L. solanacearum’ has been hampered by the difficulty in functionally characterizing the proteins of this organism, largely because they are not easily expressed and purified in standard expression systems. ‘Ca. L. solanacearum’ has a reduced genome and its proteins are predicted to be prone to instability and aggregation. Among intracellular-dwelling bacteria, chaperone proteins are conserved and overexpressed to buffer against problems in protein folding. We mimicked this approach for expressing and purifying ‘Ca. L. solanacearum’ proteins in Escherichia coli by coexpressing them with chaperones. Neither of the representative ‘Ca. L. solanacearum’ enzymes, dihydrodipicolinate synthase (key in lysine biosynthesis) and pyruvate kinase (involved in glycolysis), were overexpressed in standard E. coli expression plasmids or strains. However, soluble dihydrodipicolinate synthase was successfully coexpressed with GroEL/GroES, while soluble pyruvate kinase was successfully coexpressed with either GroEL/GroES, dnaK/dnaJ/grpE, or a trigger factor. Both enzymes, believed to be key proteins for the organism, were purified by a combination of affinity chromatography and size-exclusion chromatography. Additionally, both ‘Ca. L. solanacearum’ enzymes are active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial orthologs. This is the first study to successfully isolate and functionally characterize proteins from ‘Ca. L. solanacearum’. Thus, we provide a general strategy for characterizing its proteins, enabling new research and drug discovery programs to study and manage the pathogenicity of the organism.

‘Candidatus Liberibacter asiaticus’ is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of ‘Ca. L. asiaticus’ genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 ‘Ca. L. asiaticus’ strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that ‘Ca. L. asiaticus’ populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.