High-quality genome assembly and annotation of five bacteria isolated from the Abu Dhabi sabkha-shore region.

OBJECTIVES: Sabkhas represent polyextreme environments characterized by elevated salinity levels, intense ultraviolet (UV) radiation exposure, and extreme temperature fluctuations. In this study, we present the complete genomes of five bacterial isolates isolated from the sabkha-shore region and investigate their genomic organization and gene annotations. A better understanding of the bacterial genomic organization and genetic adaptations of these bacteria holds promise for engineering microbes with tailored functionalities for diverse industrial and agricultural applications, including bioremediation and promotion of plant growth under salinity stress conditions. DATA DESCRIPTION: We present a comprehensive genome sequencing and annotation of five bacteria (kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11) obtained from the shores of the Abu Dhabi Sabkha region. Initial bacterial identification was conducted through 16 S rDNA amplification and sequencing. Employing a hybrid genome assembly technique combining Illumina short reads (NovaSeq 6000) and Oxford Nanopore long reads (MinION), we obtained complete annotated high-quality gap-free genome sequences. The genome sizes of the kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11 isolates were determined to be 2.4 Mb, 4.1 Mb, 2.9 Mb, 5.05 Mb, and 4.1 Mb, respectively. Our analysis conclusively assigned the bacterial isolates as Staphylococcus capitis (kcgeb_sa), Bacillus spizizenii (kcgeb_sc and kcgeb_S11), Pelagerythrobacter marensis (kcgeb_sd), and Priestia aryabhattai (kcgeb_S4).

Ticarcillin degradation product thiophene acetic acid is a novel auxin analog that promotes organogenesis in tomato.

Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.

CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus).

Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.

The multifaceted roles of R2R3 transcription factor HlMYB7 in the regulation of flavonoid and bitter acids biosynthesis, development and biotic stress tolerance in hop (Humulus lupulus L.).

Hop (Humulus lupulus) biosynthesizes the highly economically valuable secondary metabolites, which include flavonoids, bitter acids, polyphenols and essential oils. These compounds have important pharmacological properties and are widely implicated in the brewing industry owing to bittering flavor, floral aroma and preservative activity. Our previous studies documented that ternary MYB-bHLH-WD40 (MBW) and binary WRKY1-WD40 (WW) protein complexes transcriptionally regulate the accumulation of bitter acid (BA) and prenylflavonoids (PF). In the present study, we investigated the regulatory functions of the R2R3-MYB repressor HlMYB7 transcription factor, which contains a conserved N-terminal domain along with the repressive motif EAR, in regulating the PF- and BA-biosynthetic pathway and their accumulation in hop. Constitutive expression of HlMYB7 resulted in transcriptional repression of structural genes involved in the terminal steps of biosynthesis of PF and BA, as well as stunted growth, delayed flowering, and reduced tolerance to viroid infection in hop. Furthermore, yeast two-hybrid and transient reporter assays revealed that HlMYB7 targets both PF and BA pathway genes and suppresses MBW and WW protein complexes. Heterologous expression of HlMYB7 leads to down-regulation of structural genes of flavonoid pathway in Arabidopsis thaliana, including a decrease in anthocyanin content in Nicotiana tabacum. The combined results from functional and transcriptomic analyses highlight the important role of HlMYB7 in fine-tuning and balancing the accumulation of secondary metabolites at the transcriptional level, thus offer a plausible target for metabolic engineering in hop.

Mechanisms of Microbial Plant Protection and Control of Plant Viruses.

Plant viral diseases are major constraints causing significant yield losses worldwide in agricultural and horticultural crops. The commonly used methods cannot eliminate viral load in infected plants. Many unconventional methods are presently being employed to prevent viral infection; however, every time, these methods are not found promising. As a result, it is critical to identify the most promising and sustainable management strategies for economically important plant viral diseases. The genetic makeup of 90 percent of viral diseases constitutes a single-stranded RNA; the most promising way for management of any RNA viruses is through use ribonucleases. The scope of involving beneficial microbial organisms in the integrated management of viral diseases is of the utmost importance and is highly imperative. This review highlights the importance of prokaryotic plant growth-promoting rhizobacteria/endophytic bacteria, actinomycetes, and fungal organisms, as well as their possible mechanisms for suppressing viral infection in plants via cross-protection, ISR, and the accumulation of defensive enzymes, phenolic compounds, lipopeptides, protease, and RNase activity against plant virus infection.

Identification and characterization of long non-coding RNA and their response against citrus bark cracking viroid infection in Humulus lupulus.

Long non-coding RNAs (lncRNAs) are a highly heterogeneous class of non-protein-encoding transcripts that play an essential regulatory role in diverse biological processes, including stress responses. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) poses a major threat to the production of Humulus lupulus (hop) plants. In this study, we systematically investigate the characteristics of the lncRNAs in hop and their role in CBCVd-infection using RNA-sequencing data. Following a stringent filtration criterion, a total of 3598 putative lncRNAs were identified with a high degree of certainty, of which 19% (684) of the lncRNAs were significantly differentially expressed (DE) in CBCVd-infected hop, which were predicted to be mainly involved in plant-pathogen interactions, kinase cascades, secondary metabolism and phytohormone signal transduction. Besides, several lncRNAs and CBCVd-responsive lncRNAs were identified as the precursor of microRNAs and predicted as endogenous target mimics (eTMs) for hop microRNAs involved in CBCVd-infection.

Establishment of CRISPR/Cas9 mediated targeted mutagenesis in hop (Humulus lupulus).

The CRISPR/Cas9-based targeted genome editing has emerged as a versatile technique, widely employed in plant genome engineering, both to decipher gene function and as an alternative to classical breeding technique for traits improvement in plants. However, to date, no such platform has been developed for hop (Humulus lupulus L.), which is an economically important crop producing valuable secondary metabolites utilized in the brewing and pharmaceutical industries. Here, we present the first report on the successful establishment of efficient CRISPR/Cas9-based genome editing using the visible endogenous marker gene phytoene desaturase (PDS) involved in carotenoid biosynthesis to demonstrate successful genome editing in hop. Agrobacterium tumefaciens-mediated transformation of in vitro generated internodal explants was used for the stable integration of constructs expressing plant codon-optimized Cas9 and a pair of co-expressed guide RNAs to target the distinct genomic sites of the PDS gene of hop. Analysis of RNA-guided genome-editing events, including mutant lines screening and homozygosity assessment using the T7 endonuclease assay showed that 33.3% of transformed plants were successfully edited at the target site, displaying albino and mosaic regenerants. Intriguingly, the detected mutations were ranges of deletions (16 bp to 39 bp) which led to disruption of the exon-intron boundary, few base substitutions, and a 1 bp insertion at 3 bp upstream of the PAM region of the target site. The decrease in chlorophyll a/b, and carotenoid content in the mutant lines further confirmed the functional disruption of the HlPDS gene. Taken together, our results demonstrate that the CRISPR/Cas9 system can precisely edit the targeted genome sequences, which may revolutionize our way to overcome some of the obstacles that have plagued the traits improvement in hop.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants.

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

Evaluation of Disease Severity and Global Transcriptome Response Induced by Citrus bark cracking viroid, Hop latent viroid, and Their Co-Infection in Hop (Humulus lupulus L.).

Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239-401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions.

Transcriptome-based mining and expression profiling of Pythium responsive transcription factors in Zingiber sp.

Transcription factors (TFs) fine-tune the host defense transcriptome in response to pathogen invasions. No information is available on Zingiber zerumbet (Zz) TFs involved in defense response against Pythium myriotylum. Here, we provide a global identification, characterization, and temporal expression profiling of Zz TFs following an incompatible interaction with P. myriotylum using a transcriptome sequencing approach. We identified a total of 903 TFs belonging to 96 families based on their conserved domains. Evolutionary analysis clustered the Zz TFs according to their phylogenetic affinity, providing glimpses of their functional diversities. High throughput expression array analysis highlighted a complex interplay between activating and repressing transcription factors in fine-tuning Zz defense response against P. myriotylum. The high differential modulation of TFs involved in cell wall fortification, lignin biosynthesis, and SA/JA hormone crosstalk allows us to envisage that this mechanism plays a central role in restricting P. myriotylum proliferation in Zz. This study lays a solid foundation and provides valuable resources for the investigation of the evolutionary history and biological functions of Zz TF genes involved in defense response.

Genome-Wide Transcriptomic Analysis Reveals Insights into the Response to Citrus bark cracking viroid (CBCVd) in Hop (Humulus lupulus L.).

Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities.

The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, (1)H NMR, (13)C NMR, (1)H-(1)H COSY, (1)H-(13)C HMBC) and Marfey's method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(L-Pro-L-Trp), cyclo(L-Leu-L-Val), cyclo(D-Pro-D-Met), and cyclo(D-Pro-D-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(L-Leu-L-Val). Cyclo(L-Leu-L-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(L-Pro-L-Trp) against S. aureus (4 µg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 µg/ml by cyclo(L-Pro-L-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete's foot, jock itch, and ringworm. The activity of cyclo(L-Pro-L-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 µg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial secondary metabolites, which may receive greater benefit as potential sources of new drugs in the pharmaceutical industry.

Genetic diversity of Phytophthora colocasiae isolates in India based on AFLP analysis.

Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro which is widely distributed in India. Inter and intra-specific genetic diversity among P. colocasiae isolates collected from same field was assessed using amplified fragment length polymorphism (AFLP) marker. Seven primer pairs produced 431 markers, of which 428 (99.2 %) were polymorphic. Considerable genetic variability was displayed by the isolates. The average value of the number of observed alleles, the number of effective alleles, mean Nei's genetic diversity, and Shannon's information index were 1.993, 1.385, 0.261, and 0.420, respectively. Analysis of molecular variance (AMOVA) showed that the majority (85 %) of the diversity were present within populations of P. colocasiae. Dendrogram based on AFLP molecular data using the unweighted pair group method with arithmetic mean (UPGMA) classified the P. colocasiae isolates into two major clusters irrespective of their geographical origin. Clustering was further supported by principle coordinate analysis. Cophenetic correlation coefficient between dendrogram and original similarity matrix was significant (r = 0.816). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed.