Development of one step colorimetric RT-LAMP assays for rapid detection of Apple mosaic virus and Prunus necrotic ringspot virus.

Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV), belonging to genus Ilarvirus, cause significant losses to rose and other plants of the family Rosaceae. They are easily transmitted through mechanical or vegetative means. In our previous study, the occurrence of ApMV and PNRSV in rose plants was reported. In this study, as a first step towards the development of a colorimetric Reverse Transcriptase - Loop Mediated Isothermal Amplification (RT-LAMP) assay, two primer sets were designed, each containing six primers (F3, B3, FIP, BIP, LF and LB) targeting the coat protein genes of ApMV and PNRSV. After incubation of RT-LAMP reaction mix at an isothermal temperature (65 °C/30 min), the amplified products were visually confirmed with the nucleic acid intercalation dye SYBR Green I and the indicator dye Hydroxy-Naphthol Blue. The developed assays were virus specific and showed no cross amplification. Their sensitivity was 103 times higher than that of the corresponding RT-PCRs. The LAMP assays developed in this study are inexpensive, rapid and reliable for the early detection of ApMV and PNRSV, and could therefore be used in plant quarantine to control the risk of their spread.

Proteometabolomic Study of Compatible Interaction in Tomato Fruit Challenged with Sclerotinia rolfsii Illustrates Novel Protein Network during Disease Progression.

Fruit is an assimilator of metabolites, nutrients, and signaling molecules, thus considered as potential target for pathogen attack. In response to patho-stress, such as fungal invasion, plants reorganize their proteome, and reconfigure their physiology in the infected organ. This remodeling is coordinated by a poorly understood signal transduction network, hormonal cascades, and metabolite reallocation. The aim of the study was to explore organ-based proteomic alterations in the susceptibility of heterotrophic fruit to necrotrophic fungal attack. We conducted time-series protein profiling of Sclerotinia rolfsii invaded tomato (Solanum lycopersicum) fruit. The differential display of proteome revealed 216 patho-stress responsive proteins (PSRPs) that change their abundance by more than 2.5-fold. Mass spectrometric analyses led to the identification of 56 PSRPs presumably involved in disease progression; regulating diverse functions viz. metabolism, signaling, redox homeostasis, transport, stress-response, protein folding, modification and degradation, development. Metabolome study indicated differential regulation of organic acid, amino acids, and carbohydrates paralleling with the proteomics analysis. Further, we interrogated the proteome data using network analysis that identified two significant functional protein hubs centered around malate dehydrogenase, T-complex protein 1 subunit gamma, and ATP synthase beta. This study reports, for the first-time, kinetically controlled patho-stress responsive protein network during post-harvest storage in a sink tissue, particularly fruit and constitute the basis toward understanding the onset and context of disease signaling and metabolic pathway alterations. The network representation may facilitate the prioritization of candidate proteins for quality improvement in storage organ.

Proteometabolomic analysis of transgenic tomato overexpressing oxalate decarboxylase uncovers novel proteins potentially involved in defense mechanism against Sclerotinia.

UNLABELLED: Oxalic acid (OA) plays dual role in fungal pathogenicity in a concentration dependent manner. While at higher concentration it induces programmed cell death leading to fungal invasion, low oxalate build resistance in plant. Although OA has been identified as a virulence determinant for rot disease caused by Sclerotinia sp., our understanding of how oxalate downregulation impart host immunity is limited. We have earlier shown that ectopic expression of oxalate decarboxylase (FvOXDC) specifically degrades OA in tomato (Solanum lycopersicum). To elucidate low oxalate regulated molecular mechanism imparting immunity, a comparative proteomics approach has been applied to E8.2-OXDC tomato fruit displaying fungal resistance. Mass spectrometric analyses identified 92 OXDC-responsive immunity related protein spots (ORIRPs) presumably associated with acid metabolism, defense signaling and endoplasmic reticulum stress. Metabolome study indicated increased abundance of some of the organic acids paralleling the proteomic analysis. Further, we interrogated the proteome data using network analysis that identified modules enriched in known and novel immunity-related prognostic proteins centered around 14-3-3, translationally controlled tumor protein, annexin and chaperonin. Taken together, our data demonstrate that low oxalate may act as metabolic and immunity determinant through translational reprogramming. BIOLOGICAL SIGNIFICANCE: Although OA plays critical role as fungal elicitor, our understanding of how oxalate downregulation by decarboxylative degradation impart immunity is limited. Our study confirms the impact of oxalate down-regulation on overall cellular physiology and provides new perspectives to study plant immunity. The network representation may facilitate the prioritization of candidate proteins for patho-stress tolerance in crop plant. These findings are of great importance for future work towards functional determination and exploitation of target proteins in crop improvement program.