Production of fluorescent antibody-labeling proteins in plants using a viral vector and the application in the detection of Acidovorax citrulli and Bamboo mosaic virus.

Serological methods are relatively convenient and simple for the detection of pathogens for front-line workers. On-site visualization of the test results plays a pivotal role in the process. However, an efficient, universal labeling agent for antibodies is needed for the development of efficient serological detection tools. In this study, a Bamboo mosaic virus (BaMV)-based viral vector was employed to express recombinant proteins, collectively designated GfED, consisting of Staphylococcus aureus Protein A domain ED (SpaED) fused to either the N- or C-terminal of an improved green florescent protein (GFP) with or without the coat protein (CP) of BaMV, efficiently in Chenopodium quinoa. The GfED in crude leaf extracts could specifically attach to IgG molecules of rabbits and mice, effectively labeling IgG with GFP, emitting green light at 506 nm when excited at 450 nm using simple, handheld equipment. To demonstrate the applicability of GfED in serological assays, we have developed a fluorescent dot blot assay for the rapid detection of Acidovorax citrulli (Ac), a bacterial pathogen of cucurbits, and BaMV, a viral pathogen of bamboos. By using the crude extracts of inoculated C. quinoa leaves expressing GfED as an IgG-labeling agent, the pathogens were easily and quickly detected through uncomplicated operations using simple equipment, with results observable by the naked eye. Examination using fluorescent microscopy and transmission electron microscopy revealed that the GfED subunits may assemble into virus-like particles, which were further involved in the formation of aggregates of GfED-antibody-antigen complexes with the potential for fluorescence signal enhancement. The results suggested that plant-expressed GfED may serve as a promising alternative of IgG-labeling agent for current serological assays.

Cross-talk between bacterial two-component systems drives stepwise regulation of flagellar biosynthesis in swarming development.

Swarming motility is a mode of bacterial movement over a solid surface driven by rotating flagella in a coordinated manner. Bacteria can use two-component system (TCS), which typically comprises a sensor kinase and a specific cognate response regulator, to properly react to environmental changes. We previously showed that the TCS RssAB suppresses flagellar biosynthesis master regulator flhDC specifically in swarming lag phase to control surface migration timing without affecting expansion rate in Serratia marcescens swarming development. Here we demonstrate that the TCS QseBC, which has been found in several human pathogens involved in flagellar and virulence regulation, has cross-talk with RssAB. We demonstrate that the phosphorylated QseB repressed flhDC expression, reducing swarming migration rate with modest effect on migration initiation. Unexpectedly, the QseC can dephosphorylate non-cognate response regulator RssB. Deletion of qseC prolonged RssAB signaling, reduced flhDC expression, and delayed migration initiation. Our data suggest that QseC is a flagellar biosynthesis activator by de-repressing RssB âˆ¼ P and QseB âˆ¼ P respectively in lag and migration phases in a stage-specific manner in swarming development.

Differential expression of pectolytic enzyme genes in Xanthomonas citri subsp. citri and demonstration that pectate lyase Pel3 is required for the formation of citrus canker.

Bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. The pectolytic enzymes produced by phytobacteria are important virulence factors involved in tissue maceration, electrolyte loss and cell death of host plants. In this study, the promoter activity of the pectolytic enzyme genes pel1, pel2, pel3, pglA, and peh-1 were investigated in Xcc XW19 strain using the ß-glucuronidase (GUS) gene as a reporter. GUS activity expressed under the control of the pel1, pel3, pglA, and peh-1 gene promoters positively correlated with bacterial growth. These gene promoters displayed high GUS activity in the presence of sodium polypectate. In addition, the four genes were induced in XVM2 minimal medium. However, only pel1 was subjected to catabolite repression by glucose. GUS activity was significantly enhanced in the XW19-derived reporter strains after they were inoculated into the leaves of Mexican lime and grapefruit, suggesting the involvement of the pel1, pel3, pglA, and peh-1 genes in XW19 pathogenesis. The pel3 promoter produced the highest GUS activity under all test conditions, whereas no GUS activity was detected using the pel2 promoter in vitro and in planta. In comparison with wild type Xcc, a pel3 mutant generated from Xcc XW19 using unmarked mutagenesis displayed reduced growth and induced smaller canker lesions on the leaves of Mexican lime, demonstrating that Pel3 of Xcc strain XW19 is a virulence factor.

The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate type III secretion system (T3SS) function and t3ss expression in Pseudomonas syringae pv. averrhoi.

To ensure the optimal infectivity on contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream of hrpG, encodes a T3SS-dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on elicitation of disease symptoms in the host and a hypersensitive response in non-host plants, and the secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild-type results in delayed HR and reduced t3ss expression. The results of protein-protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial cytoplasm, which is reduced by a single amino acid substitution at the 67th lysine residue of HrpF with alanine. Taken together, the data presented here suggest that HrpF has two roles in the assembly of a functional T3SS: one by acting as a negative regulator, possibly involved in the HrpSVG regulation circuit via binding to HrpG, and the other by stabilizing HrpA in the bacterial cytoplasm via HrpF-HrpA interaction prior to the secretion and formation of Hrp pilus on the bacterial surface.

Plant innate immunity induced by flagellin suppresses the hypersensitive response in non-host plants elicited by Pseudomonas syringae pv. averrhoi.

A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type does in non-host plants. Furthermore, the purified Pav flagellin, but not the divergent flagellin from Agrobacterium tumefaciens, is able to impair the HR induced by PA5ΔfliC. PA5Δfgt1 possessing nonglycosylated flagella behaved as its wild type in both bacterial growth in host and HR elicitation. Flagellin was infiltrated into tobacco leaves either simultaneously with flagellum-deficient HL1 or prior to the inoculation of wild type HL1, and both treatments impaired the HR induced by HL1. Moreover, the HR elicited by PA5 and PA5ΔfliC was enhanced by the addition of cycloheximide, suggesting that the flagellin is one of the PAMPs (pathogen-associated molecular patterns) contributed to induce the PAMP-triggered immunity (PTI). Taken together, the results shown in this study reveal that flagellin in Pav is capable of suppressing HR via PTI induction during an incompatible interaction.

Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase.

L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% ß-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.

Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil-body system.

Royalisin found in the royal jelly of Apis mellifera is an antimicrobial peptide (AMP). It has a molecular weight of 5.5 kDa, which contains six cysteine residues. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as two oleosin-fusion proteins for preparation of its antibodies and functional purification. The recombinant royalisin, fused with oleosin central hydrophobic domain in both N- and C-termini, was reconstituted with triacylglycerol and phospholipids to form artificial oil bodies (AOBs). The AOBs were then purified to raise the antibodies. These antibodies could recognize both the native and recombinant royalisins, but not oleosin. Another oleosin-intein S-fusion protein was purified by AOBs system, and royalisin was subsequently released from the AOBs through self-splicing of the intein. The recombinant royalisin exhibited high antibacterial activity, which suggested that it was refolded to its functional structure. These results demonstrated that AOBs system is an efficient method to functionally express and purify small AMPs. In addition, it also provides a facile platform for the production of antibodies against small peptides.

Effects of galU mutation on Pseudomonas syringae-plant interactions.

Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role in carbohydrates biosynthesis, including synthesis of lipopolysaccharides (LPS), membrane-derived oligosaccharides, and capsular polysaccharides. In this study, we characterized the galU mutant of Pseudomonas syringae pv. syringae 61 (Psy61), a necrotizing plant pathogen whose pathogenicity depends on a functional type III secretion system (T3SS), and showed that the Psy61 galU mutant had reduced biofilm formation ability, was nonmotile, and had an assembled T3SS structure but failed to elicit hypersensitive response in resistant plants and necrotic lesions in susceptible plants. Moreover, the defective LPS and other pathogen-associated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMP-triggered immunity, which severely compromised the ability of the Psy61 galU mutant to survive in planta. Our results demonstrated that the complete LPS protected plant-pathogenic bacteria from host innate immunity, similar to what was found in animal pathogens, prior to the translocation of T3S effectors and bacterial multiplication.

Unique features of Erwinia chrysanthemi (Dickeya dadantii) RA3B genes involved in the blue indigoidine production.

Erwinia chrysanthemi (Ech) RA3B produces a large amount of blue indigoidine. Using Tn5-induced mutagenesis, three indigoidine-deficient mutants were generated. Followed by library screening, a 5.8kb fragment complemented mutants for indigoidine synthesis was cloned. This fragment contains four complete open-reading frames (ORFs), pecS, pecM, idgA, and idgB, and two partial ORFs, argG, and idgC. These genes are nearly identical to those in strain Ech3937. Primer extension assays demonstrated a clear transcriptional start site prior to idgA, while no promoter preceding idgB and idgC was detected, suggesting that idgA, idgB, and idgC are organized as one transcription unit. In contrast, indAB is separated from indC in Ech3937. Interestingly, an ERIC sequence was present between idgB and idgC in place of the promoter region of the homolog indC, which may contribute to the loss of promoter activity in RA3B. Futhermore, idgB mutant displayed much lighter blue color, while indB mutant appeared white on media. Overexpression of pecS in RA3B resulted in significantly reduced indigoidine production and idgC transcript. Moreover, gel shift and luxAB reporter assays revealed that PecS specifically binds to the sequence preceding idgA and inhibits gene expression, which is consistent with the results observed in Ech3937.

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.

Identification of Pseudomonas syringae pv. syringae 61 type III secretion system Hrp proteins that can travel the type III pathway and contribute to the translocation of effector proteins into plant cells.

Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.

A chaperone-like HrpG protein acts as a suppressor of HrpV in regulation of the Pseudomonas syringae pv. syringae type III secretion system.

The cloned hrp/hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins, and many of those are assembled into a type III secretion system (TTSS) that is responsible for eliciting the hypersensitive response (HR) in non-host plants and causing diseases on host plants (Huang et al., 1995). hrpG, the second gene in the hrpC operon, encodes a 15.4 kDa cytoplasmic protein whose predicted structure is similar to SicP (E-value: 0.19), a TTSS chaperone of Salmonella typhimurium. Two non-polar hrpG mutants, Pss61-N826 and Pss61-N674, were produced to investigate the biological function of hrpG gene. Pss61-N826, generated by replacing the coding sequence of hrpG with an nptII gene lacking both the promoter and the terminator, was found to be capable of eliciting the wild-type HR; whereas Pss61-N674 generated by replacement of a terminatorless nptII gene in the hrpG coding sequence showed the delayed HR phenotype. Northern and Western blotting analyses showed that the expression of hrpZ, hrcJ and hrcQb genes residing on two different operons in Pss61-N674 was reduced due to the nptII promoter-driven constitutive expression of hrpV that codes for a negative regulator. Interestingly, a plasmid-borne hrpG can derepress the hrp expression in Pss61-N674 and in Pss61 overexpressing HrpV without decreasing the hrpV transcript. Moreover, results of yeast two-hybrid assay, pull-down assay and far Western analysis show that HrpG and HrpV interact with each other in vivo and in vitro. Additionally, HrpV interacts with a positive regulator HrpS according to analysis of a yeast two-hybrid system. Based on the results presented in this study, we propose that HrpG acts as a suppressor of the negative regulator HrpV mediated via protein-protein interaction, leading to modulation of hrp/hrc expression subsequently freeing HrpS to promote the activation of other downstream hrp/hrc genes.