Metatranscriptome analysis of symptomatic bitter apple plants revealed mixed viral infections with a putative novel polerovirus.

BACKGROUND: Next-generation Sequencing (NGS) combined with bioinformatic analyses constitutes a powerful approach for identifying and characterizing previously unknown viral genomes. In this study, leaf samples from bitter apple plants (Citrullus colocynthis (L.) Schrad) exhibiting symptoms such as dwarfing, leaf crinkling, and chlorosis were collected from the southern part of Kerman province, Iran. RESULTS: Putative infecting viruses were identified through de novo assembly of sequencing reads using various tools, followed by BLAST analysis. Complete genomes for Squash vein yellowing virus (SqVYV), Citrus-associated rhabdovirus (CiaRV), and a novel polerovirus-related strain termed Bitter apple aphid-borne yellows virus (BaABYV) were assembled and characterized. Additionally, a partial genome for Watermelon mosaic virus (WMV) was assembled. The genomic organization of the BaABYV was determined to be 5'-ORF0-ORF1-ORF1,2-ORF3a-ORF3-ORF3,5-ORF4-3'. Amino acid sequence identities for inferred proteins (P0 and P1, P1,2) with known poleroviruses were found to be the 90% species delineation limit, implying that BaABYV should be considered a new member of the genus Polerovirus. Recombination events were observed in the BaABYV and WMV strains; such events were not found in the CiaRV strain. CONCLUSIONS: Molecular evidence from this study suggests that C. colocynthis is a reservoir host of several plant viruses. Among them, BaABYV is proposed as a new member of the genus Polerovirus. Furthermore, the CiaRV strain has been reported for the first time from Iran.

Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

Analysis of the Citrullus colocynthis transcriptome during water deficit stress.

Citrullus colocynthis is a very drought tolerant species, closely related to watermelon (C. lanatus var. lanatus), an economically important cucurbit crop. Drought is a threat to plant growth and development, and the discovery of drought inducible genes with various functions is of great importance. We used high throughput mRNA Illumina sequencing technology and bioinformatic strategies to analyze the C. colocynthis leaf transcriptome under drought treatment. Leaf samples at four different time points (0, 24, 36, or 48 hours of withholding water) were used for RNA extraction and Illumina sequencing. qRT-PCR of several drought responsive genes was performed to confirm the accuracy of RNA sequencing. Leaf transcriptome analysis provided the first glimpse of the drought responsive transcriptome of this unique cucurbit species. A total of 5038 full-length cDNAs were detected, with 2545 genes showing significant changes during drought stress. Principle component analysis indicated that drought was the major contributing factor regulating transcriptome changes. Up regulation of many transcription factors, stress signaling factors, detoxification genes, and genes involved in phytohormone signaling and citrulline metabolism occurred under the water deficit conditions. The C. colocynthis transcriptome data highlight the activation of a large set of drought related genes in this species, thus providing a valuable resource for future functional analysis of candidate genes in defense of drought stress.

Citrullus colocynthis NAC transcription factors CcNAC1 and CcNAC2 are involved in light and auxin signaling.

KEY MESSAGE: Two novel NAC transcription factors from C itrullus colocynthis implicated in light and auxin signaling pathway. NAC transcription factors (NAM, ATAF1, 2, CUC2) have multiple functions in plant growth and development. Two NACs, CcNAC1 and CcNAC2, were recently identified in the highly drought-tolerant cucurbit species, Citrullus colocynthis. This study examines the functional role of these genes under different qualities of light based on the in silico analysis of the CcNAC1 and CcNAC2 promoters that revealed the presence of several light-associated motifs. The impact of both light and auxin on CcNAC1 and CcNAC2 expression was examined in C. colocynthis leaves, and using reporter (pCcNAC1, 2::GUS) lines in Arabidopsis. Furthermore, the effects of constitutive overexpression (OE-CcNAC1, 2) in Arabidopsis were also examined under a range of conditions to confirm reporter line linkages. White, blue, red, and far-red light treatments resulted in similar patterns of quantitative changes in CcNAC1and CcNAC2 expression in both species, with the highest transcript increases following red light. Photomorphogenic changes in Arabidopsis hypocotyls were correlated with gene transcript levels. In the absence of light, hypocotyls of OE-CcNAC1/CcNAC2 lines were significantly longer as compared to WT. The addition of exogenous auxin (+IAA) to growth medium also resulted in changes to the hypocotyl lengths of overexpression lines and spatiotemporal reporter line changes in seedlings. Our data suggest that CcNAC1, 2 might be functionally important in the light signaling pathway, and appear connected to the hormone auxin. This is the first study to indicate that NAC genes might play a role in both light and auxin signaling pathways.