Biocontrol of sheath blight of rice (Oryza sativa L.) through alteration in expression dynamics of candidate effector genes of Rhizoctonia solani AG1-IA during pathogenesis.

In genome analyses of Rhizoctonia solani AG1-IA causing sheath blight (ShB) of rice, many genes were identified to have a hypothetical role in pathogenesis. To understand their roles in pathogenesis, their expressions during fungal infection were studied. An aggressive R. solani strain, RIRS-K, was first identified among six isolates, RIRS-K, RIRS-17, RIRS-S, RIRS-T, RIRS-MU and RIRS-FD, for inducing a maximum relative lesion height (RLH) of 32.7% on a ShB susceptible cultivar, Pusa Basmati-1. Hypothetical pathogenicity genes (52 nos) identified by in silico analyses of the publicly available genomic database of the pathogen strain were evaluated in Pathogen-Host Interaction (PHI) blast and RIRS-K. Though PHI blast identified 26 genes as potential ones, only 8 were constitutively expressive in RIRS-K cultured in a minimal broth. Among them, only expressions of AG1IA_06195, AG02692, AG04508, and AG05730 were induced in the rice plant inoculated with RIRS-K and, hence, were identified as the candidate ones. The candidate genes were highly expressed in the aggressive strain (RIRS-K) in comparison to the less aggressive one (RIRS-17). In further testing of their expressions in the highly aggressive fungal strain, RIRS-K infecting PB-1 pre-colonized by a potent biocontrol consortium comprising of Bacillus subtilis (S17TH), Pseudomonas putida (TEPF-Sungal-1), and Trichoderma harzianum (S17TH), the disease scoring and gene expression studies indicated that the candidate genes were downregulated. The studies, therefore, speculated that these genes might play a role in pathogen aggressiveness and ShB development.

Metabolomic analysis of sheath blight disease of rice (Oryza sativa L.) induced by Rhizoctonia solani phytotoxin.

AIM: To understand the mechanism of necrosis incited by a host-selective phytotoxin designated as Rhizoctonia solani toxin (RST) identified to be a potential pathogenic factor of R. solani AG1 IA, causing sheath blight (ShB) of rice. METHODS AND RESULTS: The metabolomic changes induced by the phytotoxic metabolite in a ShB susceptible rice cultivar were elucidated by gas chromatography-mass spectrometry analysis and compared with that of the pathogen to identify rice metabolites targeted by the phytotoxin. The profiles of about 29 metabolites with various physiological roles in rice plants have been identified worldwide. Unsupervised and supervised multivariate chemometrics (principal component analysis and partial least squares-discriminant analysis) and cluster (Heat maps) analyses were used to compare the metabolites obtained from chemical profiles of the treatments with sterile distilled water (SDW) control. The results indicated that the rice plant expressed more metabolites in response to the pathogen than the phytotoxin and was lowest in SDW control. The key metabolites expressed in rice in response to the treatments were investigated by the variable importance in projection (VIP) analysis using p < 0.05 VIP >15. The analysis identified 7 and 11 upregulating metabolites in the phytotoxin and the pathogen treatments, respectively, compared to the untreated control. Among the phytotoxin-treated and the pathogen inoculated samples, the phytotoxin-treated sample recorded upregulation of six metabolites, whereas nine metabolites were upregulated in the pathogen-inoculated samples. These upregulating metabolites are speculated for the necrotic symptoms characteristic to both the phytotoxin and pathogen. In this analysis, hexadecanoic acid and dotriacontane were highly expressed metabolites specific to the phytotoxin and pathogen-treated samples, respectively. Besides upregulation, the metabolites also have a VIP score of >1.5 and hence fulfilled the criteria of classifying them as reliable potential biomarkers. In the pathway analysis, hexadecanoic acid and dotriacontane were identified to be involved in several important biosynthetic pathways of rice, such as the biosynthesis of saturated fatty acid and unsaturated fatty acids cutin, suberin and wax. CONCLUSIONS: The study concludes that though certain metabolites induced by the phytotoxin in the susceptible variety during necrosis shares with that of the pathogen, the identification of metabolites specific to the phytotoxin in comparison to the pathogenic and SDW controls indicated that the phytotoxin modulates the host metabolism differently and hence can be a potential pathogenicity factor of the ShB fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to lack of knowledge on the pathway genes of RST and in the absence of an ShB-resistant variety, understanding differentially expressed metabolic changes induced in the susceptible variety by the phytotoxin in comparison to that of the pathogenic and uninoculated controls enables us to identify the key metabolite changes during the ShB infection. Such metabolomic changes can further be used to infer gene functions for exploitation in ShB control.

Genome-Wide Identification and Characterisation of Cytokinin-O-Glucosyltransferase (CGT) Genes of Rice Specific to Potential Pathogens.

Cytokinin glucosyltransferases (CGTs) are key enzymes of plants for regulating the level and function of cytokinins. In a genomic identification of rice CGTs, 41 genes with the plant secondary product glycosyltransferases (PSPG) motif of 44-amino-acid consensus sequence characteristic of plant uridine diphosphate (UDP)-glycosyltransferases (UGTs) were identified. In-silico physicochemical characterisation revealed that, though the CGTs belong to the same subfamily, they display varying molecular weights, ranging from 19.6 kDa to 59.7 kDa. The proteins were primarily acidic (87.8%) and hydrophilic (58.6%) and were observed to be distributed in the plastids (16), plasma membrane (13), mitochondria (5), and cytosol (4). Phylogenetic analysis of the CGTs revealed that their evolutionary relatedness ranged from 70-100%, and they aligned themselves into two major clusters. In a comprehensive analysis of the available transcriptomics data of rice samples representing different growth stages only the CGT, Os04g25440.1 was significantly expressed at the vegetative stage, whereas 16 other genes were highly expressed only at the reproductive growth stage. On the contrary, six genes, LOC_Os07g30610.1, LOC_Os04g25440.1, LOC_Os07g30620.1, LOC_Os04g25490.1, LOC_Os04g37820.1, and LOC_Os04g25800.1, were significantly upregulated in rice plants inoculated with Rhizoctonia solani (RS), Xoo (Xanthomonas oryzae pv. oryzae) and Mor (Magnaporthe oryzae). In a qRT-PCR analysis of rice sheath tissue susceptible to Rhizoctonia solani, Mor, and Xoo pathogens, compared to the sterile distilled water control, at 24 h post-infection only two genes displayed significant upregulation in response to all the three pathogens: LOC_Os07g30620.1 and LOC_Os04g25820.1. On the other hand, the expression of genes LOC_Os07g30610.1, LOC_Os04g25440, LOC_Os04g25490, and LOC_Os04g25800 were observed to be pathogen-specific. These genes were identified as the candidate-responsive CGT genes and could serve as potential susceptibility genes for facilitating pathogen infection.

Genome-Wide Analysis of Cytochrome P450s of Alternaria Species: Evolutionary Origin, Family Expansion and Putative Functions.

Cytochrome P450s are a group of monooxygenase enzymes involved in primary, secondary and xenobiotic metabolisms. They have a wide application in the agriculture sector where they could serve as a target for herbicides or fungicides, while they could function in the pharmaceutical industry as drugs or drugs structures or for bioconversions. Alternaria species are among the most commonly encountered fungal genera, with most of them living as saprophytes in different habitats, while others are parasites of plants and animals. This study was conducted to elucidate the diversity and abundance, evolutionary relationships and cellular localization of 372 cytochrome P450 in 13 Alternaria species. The 372 CYP proteins were phylogenetically clustered into ten clades. Forty (40) clans and seventy-one (71) cyp families were identified, of which eleven (11) families were found to appear in one species each. The majority of the CYP proteins were located in the endomembrane system. Polyketide synthase (PKS) gene cluster was the predominant secondary metabolic-related gene cluster in all the Alternaria species studied, except in A. porriof, where non-ribosomal peptide synthetase genes were dominant. This study reveals the expansion of cyps in these fungal genera, evident in the family and clan expansions, which is usually associated with the evolution of fungal characteristics, especially their lifestyle either as parasites or saprophytes, with the ability to metabolize a wide spectrum of substrates. This study can be used to understand the biology, physiology and toxigenic potentials of P450 in these fungal genera.

Robust Profiling of Cytochrome P450s (P450ome) in Notable Aspergillus spp.

Cytochrome P450s (P450ome) constitute an extended superfamily group of heme-thiolate enzymes identified in all biological domains. P450omes play a critical role in the oxidation of steroids and fatty acids, xenobiotic degradation of hydrophobic compounds, biosynthesis of hormones, and primary and secondary metabolism in organisms. Aspergillus species are among the most economically important fungal organisms in human medicine, industry, and agriculture worldwide. Exploring insight on the genome-wide annotations of cytochrome P450s in Aspergillus species is necessary for their biosynthetic applications. In this present study, we report the identification of 306 cytochrome P450s and their robust profiling in eight notable Aspergillus species (A. carbonarius, A. clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. oryzae, and A. terreus). Based on the evolutionary relationship, the Aspergillus P450s families clustered into 15 clades, with clades V, I, and XIII recording higher percentages (17.3%, 15.00%, and 14.71%, respectively) of Cyp families. Cyps were classified into 120 families 64 clans, and their putative functions were also elucidated. P450s were predicted to be located in 13 subcellular components, but the endoplasm reticulum was the dominant location across the eight Aspergillus species. Cyps genes of Aspergillus species were associated with seven secondary metabolism-related gene clusters. Elucidating the genome-wide annotations of P450s enzymes in Aspergillus species will form vital potential biotechnological tools that could be harnessed for industrial, pharmaceutical, and agricultural use.

Base Editing in Plants: Applications, Challenges, and Future Prospects.

The ability to create targeted modifications in the genomes of plants using genome editing technologies has revolutionized research in crop improvement in the current dispensation of molecular biology. This technology has attracted global attention and has been employed in functional analysis studies in crop plants. Since many important agronomic traits are confirmed to be determined by single-nucleotide polymorphisms, improved crop varieties could be developed by the programmed and precise conversion of targeted single bases in the genomes of plants. One novel genome editing approach which serves for this purpose is base editing. Base editing directly makes targeted and irreversible base conversion without creating double-strand breaks (DSBs). This technology has recently gained quick acceptance and adaptation because of its precision, simplicity, and multiplex capabilities. This review focuses on generating different base-editing technologies and how efficient they are in editing nucleic acids. Emphasis is placed on the exploration and applications of these base-editing technologies to enhance crop production. The review also highlights the drawbacks and the prospects of this new technology.