Chunxia Wang

Transient gene expression is a powerful tool to study gene function in plants. In citrus, transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than . Here we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of effector AvrGF1. To further expand the availa...

Bacterial two-component regulatory systems (TCS) are common components of complex regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI controls exopolysaccharide succinoglycan production and flagellum biosynthesis. Although this system plays a crucial role in establishing the symbiosis between S. meliloti and its host plant, it is not well characterized. Attempts to generate complete loss-of-function mutations in either exoS or chvI in S. meliloti have been unsuccessful; thus, it was previously suggested that exoS or chvI are essential genes for bacterial cell growth. We constructed a chvI mutant by completely deleting the open reading frame encoding this gene. The mutant strain failed to grow on complex medium, exhibited lower tolerance to acidic condition, produced significantly less poly-3-hydroxybutyrate than the wild type, was hypermotile, and exhibited an altered lipopolysaccharide profile. In addition, this mutant was defective in symbiosis with Medi...

Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia-legume symbiosis are not fully understood (1, 2, 3). Bacteroids with determinate nodules often accumulate high levels of PHB (up to 70% dry weight) (1). In contrast, bacteroids within indeterminate nodules do not accumulate PHB. In the symbiosis between S. meliloti and alfalfa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild type (4, 5, 6), but are less competitive than wild type strain (5). There are two glycogen synthase-encoding genes in S. meliloti, glgA1 in a cluster of other glycogen synthesis pathway genes on the chromosome, and glgA2 on megaplasmid pSymb. Recently, it has been shown in Rhizobium tropici with Phaseolus vulgaris that there may be a link between glycogen synthase deficiency, decreased exopolysaccharide, and increased symbiotic performance (2). However, the reason for the increased symbiotic efficiency of the glgA mutant is uncertain (3). To determine the roles these compounds may play in the symbiotic process and in the overall physiology of the organism in the free-living and bacteroid states, mutants unable to synthesize PHB and/or glycogen were constructed. A glgA1 mutation was constructed by in-frame deletion, preserving the expression of the downstream pgm gene, while a glgA2 mutation was constructed by disruption of the gene with the Sp omega cassette (7). A pre-existing Tn5-generated mutation of the PHB synthase encoding gene phbC (8) was combined with glgA1 and glgA2 mutations to make all combinations of double mutants, and the triple mutant (Table 1). PHB was not detectable in free-living cells of any mutant containing the phbC mutation; glycogen was not detectable in any of the mutants containing the glgA1 mutation. The production of PHB decreased significantly in the glgA1 mutant (Rm11479), and the glgA double mutant (Rm11482). The production of glycogen increased significantly in the phbC mutant (Rm11105) in high carbon ratio media. Exopolysaccharide (EPS) was not detected in any of the mutants containing the phbC mutation, while the glgA double mutant (Rm11482) produced much more EPS in MOPS medium compared to the wild type (Rm1021), glgA1

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and u...

Funded by the National Institute of Allergy and Infectious Diseases, the Pathosystems Resource Integration Center (PATRIC) is a genomics-centric relational database and bioinformatics resource designed to assist scientists in infectious-disease research. Specifically, PATRIC provides scientists with (i) a comprehensive bacterial genomics database, (ii) a plethora of associated data relevant to genomic analysis, and (iii) an extensive suite of computational tools and platforms for bioinformatics analysis. While the primary aim of PATRIC is to advance the knowledge underlying the biology of human pathogens, all publicly available genome-scale data for bacteria are compiled and continually updated, thereby enabling comparative analyses to reveal the basis for differences between infectious free-living and commensal species. Herein we summarize the major features available at PATRIC, dividing the resources into two major categories: (i) organisms, genomes, and comparative genomics and (...