iTRAQ-based proteomic analyses of the plant-pathogenic bacterium Acidovorax citrulli during entrance into and resuscitation from the viable but nonculturable state.

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) disease, infects cucurbit crops including watermelon and melon. This bacterium can enter the viable but nonculturable (VBNC) state following exposure to copper sulfate. Moreover, copper-induced VBNC A. citrulli cells can be resuscitated by EDTA. In this study, isobaric tag for relative and absolute quantification (iTRAQ) was used to compare protein profiles of VBNC cells, resuscitated cells at different stages and log-phase cells of the A. citrulli model strain AAC00-1. A total of 2672 proteins were identified, with 60 being differentially abundant in VBNC cells compared with log-phase cells, and 469 being differentially abundant in resuscitated cells compared with VBNC cells. Proteins involved in the arginine and proline metabolism pathway and degradation of aromatic compounds could be important for the VBNC cells. In the early resuscitation process, proteins associated with carbon metabolism, and degradation of naphthalene and aromatic compounds were significantly enriched, while proteins involved in oxidative phosphorylation, bacterial chemotaxis, ABC transporters and quorum sensing were significantly enriched at the late resuscitation stages. This is the first study reporting thorough protein profile analyses of VBNC and resuscitating cells of a plant-pathogenic bacterium. BIOLOGICAL SIGNIFICANCE: The VBNC state is a dormant-like condition that was reported to occur in many bacterial species, upon facing a variety of environmental stresses. Acidovorax citrulli is a seed borne pathogenic bacterium that threatens cucurbit production worldwide. Moreover, A. citrulli can enter into the VBNC state after treatment of copper sulfate, thus increasing its survival and dissemination probabilities. This study enriches our understanding of the mechanisms of entrance into and resuscitation from the VBNC state of this important plant-pathogenic bacterium. This knowledge could be exploited in the future to develop novel approaches to interfere with these processes, thus contributing to a more efficient management of this pathogen. In a broader perspective, the knowledge emerging from this study has implications to the general understanding of the VBNC state in bacteria.

Evaluation of suitable reference genes for normalization of quantitative reverse transcription PCR analyses in Clavibacter michiganensis.

Clavibacter michiganensis, the causal agent of bacterial canker of tomato, is a Gram-positive bacterium and a model for studying plant diseases. The real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay is widely used to quantify gene expression in plant pathogenic bacteria. However, accurate quantification of gene expression requires stably expressed reference genes that are consistently expressed during the experimental conditions of interest. The use of inappropriate reference genes leads to a misinterpretation of gene expression data and false conclusions. In current study, we empirically assessed the expression stability of six housekeeping genes (gyrB, rpoB, tufA, bipA, gapA, and pbpA) of C. michiganensis under five experimental conditions using two algorithms, geNorm and NormFinder. C. michiganensis expressed gyrB, bipA, and gapA stably when growing in nutrient-rich broth (TBY broth and modified M9 broth). We concluded that pbpA, tufA, and gyrB were suitable reference genes in C. michiganensis-tomato interaction studies. We also recommended bipA and rpoB to be used to study bacterial gene expression under nutrient-poor conditions. Finally, gyrB, pbpA, and rpoB can be used to normalize the quantification of C. michiganensis gene expression while the bacterium is in the viable but nonculturable (VBNC) state. This study identified the most suitable reference genes depending on the experimental conditions for calibrating real-time qRT-PCR analyses of C. michiganensis and will be useful in studies that seek to understand the molecular interactions between C. michiganensis and tomato.

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (108 CFU mL-1) was induced by 50 µM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 µM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 103 to 107 CFU mL-1. The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.

Development and utilization of a new chemically-induced soybean library with a high mutation density .

Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate (EMS)-induced soybean (Glycine max) population, consisting of 21,600 independent M2 lines, was developed. Over 1,000 M4 (5) families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll (Chl) content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to MinnGold, where a different single nucleotide polymorphism variation in the Mg-chelatase subunit gene (ChlI1a) results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker-assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.