Characterization and development of transcriptome-derived novel EST-SSR markers to assess genetic diversity in Chaetomium globosum.

Chaetomium globosum Kunze, an internationally recognized biocontrol fungus. It mycoparasitizes various plant pathogens and produce antifungal metabolites to suppress the growth of pathogenic fungi. Lack of detailed genome level diversity studies has delimited the development and utilization of potential C. globosum strains. The present study was taken to reveal the distribution, identification, and characterization of expressed sequence tag-simple sequence repeats (EST-SSRs) in C. globosum. RNA-Seq experiment was performed for C. globosum potential isolate Cg2 (AY429049) using Illumina HiSeq 2500. Reference-guided de novo assembly yielded 45,582 transcripts containing 27,957 unigenes. We generated a new set of 8485 EST-SSR markers distributed in 5908 unigene sequences with one SSR locus distribution density per 6.1 kb. Six distinct classes of SSR repeat motifs were identified. The most abundant were mononucleotide repeats (51.67%), followed by tri-nucleotides (36.61%). Out of 5034 EST-SSR primers, 50 primer pairs were selected and validated for the polymorphic study of 15 C. globosum isolates. Twenty-two SSR markers showed average genetic polymorphism among C. globosum isolates. The number of alleles (Na) per marker ranges from 2 to 4, with a total of 74 alleles detected for 22 markers with a mean polymorphism information content (PIC) value of 0.4. UPGMA hierarchical clustering analysis generated three main clusters of C. globosum isolates and exhibited a lower similarity index range from 0.59 to 0.85. Thus, the newly developed EST-SSR markers could replace traditional methods for determining diversity. The study will also enhance the genomic research in C. globosum to explore its biocontrol potential against phytopathogens. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03794-7.

Integration of miRNA dynamics and drought tolerant QTLs in rice reveals the role of miR2919 in drought stress response.

To combat drought stress in rice, a major threat to global food security, three major quantitative trait loci for 'yield under drought stress' (qDTYs) were successfully exploited in the last decade. However, their molecular basis still remains unknown. To understand the role of secondary regulation by miRNA in drought stress response and their relation, if any, with the three qDTYs, the miRNA dynamics under drought stress was studied at booting stage in two drought tolerant (Sahbaghi Dhan and Vandana) and one drought sensitive (IR 20) cultivars. In total, 53 known and 40 novel differentially expressed (DE) miRNAs were identified. The primary drought responsive miRNAs were Osa-MIR2919, Osa-MIR3979, Osa-MIR159f, Osa-MIR156k, Osa-MIR528, Osa-MIR530, Osa-MIR2091, Osa-MIR531a, Osa-MIR531b as well as three novel ones. Sixty-one target genes that corresponded to 11 known and 4 novel DE miRNAs were found to be co-localized with the three qDTYs, out of the 1746 target genes identified. We could validate miRNA-mRNA expression under drought for nine known and three novel miRNAs in eight different rice genotypes showing varying degree of tolerance. From our study, Osa-MIR2919, Osa-MIR3979, Osa-MIR528, Osa-MIR2091-5p and Chr01_11911S14Astr and their target genes LOC_Os01g72000, LOC_Os01g66890, LOC_Os01g57990, LOC_Os01g56780, LOC_Os01g72834, LOC_Os01g61880 and LOC_Os01g72780 were identified as the most promising candidates for drought tolerance at booting stage. Of these, Osa-MIR2919 with 19 target genes in the qDTYs is being reported for the first time. It acts as a negative regulator of drought stress tolerance by modulating the cytokinin and brassinosteroid signalling pathway.

The chromosome-scale genome assembly of cluster bean provides molecular insight into edible gum (galactomannan) biosynthesis family genes.

Cluster bean (Cyamopsis tetragonoloba (L.) Taub 2n = 14, is commonly known as Guar. Apart from being a vegetable crop, it is an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan. Here, we are reporting a chromosome-scale reference genome assembly of a popular cluster bean cultivar RGC-936, by combining sequencing data from Illumina, 10X Genomics, Oxford Nanopore technologies. An initial assembly of 1580 scaffolds with an N50 value of 7.12 Mb was generated and these scaffolds were anchored to a high density SNP linkage map. Finally, a genome assembly of 550.31 Mb (94% of the estimated genome size of ~ 580 Mb (through flow cytometry) with 58 scaffolds was obtained, including 7 super scaffolds with a very high N50 value of 78.27 Mb. Phylogenetic analysis using single copy orthologs among 12 angiosperms showed that cluster bean shared a common ancestor with other legumes 80.6 MYA. No evidence of recent whole genome duplication event in cluster bean was found in our analysis. Further comparative transcriptomics analyses revealed pod-specific up-regulation of genes encoding enzymes involved in galactomannan biosynthesis. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted improvement of cluster bean.

Meta-Analysis of Microarray Data and Their Utility in Dissecting the Mapped QTLs for Heat Acclimation in Rice.

In the current global warming scenario, it is imperative to develop crops with improved heat tolerance or acclimation, for which knowledge of major heat stress-tolerant genes or genomic regions is a prerequisite. Though several quantitative trait loci (QTLs) for heat tolerance have been mapped in rice, candidate genes from these QTLs have not been reported yet. The meta-analysis of microarray datasets for heat stress in rice can give us a better genomic resource for the dissection of QTLs and the identification of major candidate genes for heat stress tolerance. In the present study, a database, RiceMetaSys-H, comprising 4227 heat stress-responsive genes (HRGs), was created using seven publicly available microarray datasets. This included in-house-generated microarray datasets of Nagina 22 (N22) and IR64 subjected to 8 days of heat stress. The database has provisions for searching the HRGs through genotypes, growth stages, tissues, and physical intervals in the genome, as well as Locus IDs, which provide complete information on the HRGs with their annotations and fold changes, along with the experimental material used for the analysis. The up-regulation of genes involved in hormone biosynthesis and signalling, sugar metabolism, carbon fixation, and the ROS pathway were found to be the key mechanisms of enhanced heat tolerance. Integrating variant and expression analysis, the database was used for the dissection of the major effect of QTLs on chromosomes 4, 5, and 9 from the IR64/N22 mapping population. Out of the 18, 54, and 62 genes in these three QTLs, 5, 15, and 12 genes harboured non-synonymous substitutions. Fifty-seven interacting genes of the selected QTLs were identified by a network analysis of the HRGs in the QTL regions. Variant analysis revealed that the proportion of unique amino acid substitutions (between N22/IR64) in the QTL-specific genes was much higher than the common substitutions, i.e., 2.58:0.88 (2.93-fold), compared to the network genes at a 0.88:0.67 (1.313-fold) ratio. An expression analysis of these 89 genes showed 43 DEGs between IR64/N22. By integrating the expression profiles, allelic variations, and the database, four robust candidates (LOC_Os05g43870, LOC_Os09g27830, LOC_Os09g27650, andLOC_Os09g28000) for enhanced heat stress tolerance were identified. The database thus developed in rice can be used in breeding to combat high-temperature stress.

Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin.

Kinnow (Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I-VII). The ovules of stage III fruits (>21-25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L-1 and malt extract (ME) 1,000 mg L-1. Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA3) 2.0 mg L-1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L-1 + spermidine 100 mg L-1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.

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.

Understanding the Dynamics of Blast Resistance in Rice-Magnaporthe oryzae Interactions.

Rice is a global food grain crop for more than one-third of the human population and a source for food and nutritional security. Rice production is subjected to various stresses; blast disease caused by Magnaporthe oryzae is one of the major biotic stresses that has the potential to destroy total crop under severe conditions. In the present review, we discuss the importance of rice and blast disease in the present and future global context, genomics and molecular biology of blast pathogen and rice, and the molecular interplay between rice-M. oryzae interaction governed by different gene interaction models. We also elaborated in detail on M. oryzae effector and Avr genes, and the role of noncoding RNAs in disease development. Further, rice blast resistance QTLs; resistance (R) genes; and alleles identified, cloned, and characterized are discussed. We also discuss the utilization of QTLs and R genes for blast resistance through conventional breeding and transgenic approaches. Finally, we review the demonstrated examples and potential applications of the latest genome-editing tools in understanding and managing blast disease in rice.

Identification of major candidate genes for multiple abiotic stress tolerance at seedling stage by network analysis and their validation by expression profiling in rice (Oryza sativa L.).

A wealth of microarray and RNA-seq data for studying abiotic stress tolerance in rice exists but only limited studies have been carried out on multiple stress-tolerance responses and mechanisms. In this study, we identified 6657 abiotic stress-responsive genes pertaining to drought, salinity and heat stresses from the seedling stage microarray data of 83 samples and used them to perform unweighted network analysis and to identify key hub genes or master regulators for multiple abiotic stress tolerance. Of the total 55 modules identified from the analysis, the top 10 modules with 8-61 nodes comprised 239 genes. From these 10 modules, 10 genes common to all the three stresses were selected. Further, based on the centrality properties and highly dense interactions, we identified 7 intra-modular hub genes leading to a total of 17 potential candidate genes. Out of these 17 genes, 15 were validated by expression analysis using a panel of 4 test genotypes and a pair of standard check genotypes for each abiotic stress response. Interestingly, all the 15 genes showed upregulation under all stresses and in all the genotypes, suggesting that they could be representing some of the core abiotic stress-responsive genes. More pertinently, eight of the genes were found to be co-localized with the stress-tolerance QTL regions. Thus, in conclusion, our study not only provided an effective approach for studying abiotic stress tolerance in rice, but also identified major candidate genes which could be further validated by functional genomics for abiotic stress tolerance. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03182-7.

Draft genome sequence of 'Candidatus Phytoplasma australasia', strain SS02 associated with sesame phyllody disease.

'Candidatus Phytoplasma' is an uncultivated, intracellular bacterial plant pathogen transmitted by phloem-feeding insect vectors. Among the group of phytoplasmas, the Peanut Witches' Broom or 16SrII group of phytoplasmas associated with various diseases cause severe crop losses every year in India. The 'Ca. Phytoplasma sp.' strain SS02 was associated with phyllody disease of sesame plants collected from New Delhi. The genome sequence of strain SS02 was obtained using its genomic DNA enrichment and hybrid assembly of sequences generated on Illumina and Oxford Nanopore Technologies MinION platforms. The hybrid assembly strategy generated a draft genome with 60 contigs totaling 553,228 bp of length with more than 400 × depth coverage and 95.21% of the estimated completeness. The SS02 genome draft sequence contains 465 protein-coding genes, 17 tRNA genes, and 3 rRNA genes. The availability of this draft genome also provided a foundation for genome-scale genotypic analyses.

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.

Corrigendum: Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium.

[This corrects the article DOI: 10.3389/fpls.2021.748013.].

Identification of novel resources for panicle blast resistance from wild rice accessions and mutants of cv. Nagina 22 by syringe inoculation under field conditions.

Panicle blast is the most severe type of rice blast disease. Screening of rice genotypes for panicle blast resistance at the field level requires an efficient and robust method of inoculation. Here, we standardized a method that can be utilized for both small- and large-scale screening and assessment of panicle blast infection and disease reaction. The method involves inoculation of Magnaporthe oryzae spore culture in the neck of the rice panicle using a syringe and covering the inoculation site with wet cotton wrapped with aluminum foil to provide the required humidity for spore germination. The method was standardized using panicle blast-resistant cv. Tetep and susceptible cv. HP2216 inoculated with Mo-ni-025 isolate of M. oryzae. The method was evaluated at phenotypic as well as molecular level by expression analysis of disease responsive pathogenesis-related (PR) genes. We found this method simple, robust, reliable, and highly efficient for screening of large germplasm sets of rice for panicle blast. This was validated by screening the wild rice germplasm for panicle blast response in the field using three M. oryzae strains and subsequently with the most virulent strain in 45 EMS-induced mutants of Nagina 22 shortlisted based on field screening in a blast hotspot region. We identified five novel blast disease-resistant wild rice genotypes and 15 Nagina 22 mutants that can be used in breeding programmes.

Overexpression of a DUF740 family gene (LOC_Os04g59420) imparts enhanced climate resilience through multiple stress tolerance in rice.

Functional characterization of stress-responsive genes through the analysis of transgenic plants is a standard approach to comprehend their role in climate resilience and subsequently exploit them for sustainable crop improvement. In this study, we investigated the function of LOC_Os04g59420, a gene of DUF740 family (OsSRDP-Oryza sativa Stress Responsive DUF740 Protein) from rice, which showed upregulation in response to abiotic stress in the available global expression data, but is yet to be functionally characterized. Transgenic plants of the rice OsSRDP gene, driven by a stress-inducible promoter AtRd29A, were developed in the background of cv. Pusa Sugandh 2 (PS2) and their transgene integration and copy number were confirmed by molecular analysis. The three independent homozygous transgenic plants (AtRd29A::OsSRDP rice transformants) showed better resilience to drought, salinity, and cold stresses, but not heat stress, as compared to the non-transformed PS2, which corresponded with their respective relative transcript abundance for OsSRDP. Transgenic plants maintained higher RWC, photosynthetic pigments, and proline accumulation under drought and salinity stresses. Furthermore, they exhibited less accumulation of reactive oxygen species (ROS) than PS2 under drought stress, as seen from the transcript abundance studies of the ROS genes. Under cold stress, OsSRDP transgenic lines illustrated minimal cell membrane injury compared to PS2. Additionally, the transgenic plants showed resistance to a virulent strain of rice blast fungus, Magnaporthe oryzae (M. oryzae). The promoter analysis of the gene in N22 and PS2 revealed the presence of multiple abiotic and biotic stress-specific motif elements supporting our observation on multiple stress tolerance. Based on bioinformatics studies, we identified four potential candidate interaction partners for LOC_Os04g59420, of which two genes (LOC_Os05g09640 and LOC_Os06g50370) showed co-expression under biotic and drought stress along with OsSRDP. Altogether, our findings established that stress-inducible expression of OsSRDP can significantly enhance tolerance to multiple abiotic stresses and a biotic stress.

Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium.

Though the vascular wilt of tomato caused by the species of Fusarium is globally reported to be a complex disease in certain countries, for example, India, our studies indicated that the disease is caused by either Fusarium oxysporum f. spp. lycopersici (Fol) or Fusarium solani (FS) with the Fol being widely prevalent. In assessing the genetic diversity of 14 Fol strains representing the four Indian states by the unweighted pair group method with arithmetic averaging using Inter Simple Sequence Repeat (ISSR) amplicons, the strains distinguished themselves into two major clusters showing no correlation with their geographic origin. In pot experiments under polyhouse conditions, the seed dressing and soil application of a talc-based formulation of a biocontrol treatment, TEPF-Sungal-1 (Pseudomonas putida) + S17TH (Trichoderma harzianum) + CG-A (Chaetomium globosum), which inhibited Fol, was equally effective like the cell suspensions and was even better than the fungicidal mixture (copper oxychloride-0.25% + carbendazim-0.1%) in promoting the crop growth (52.3%) and reducing vascular wilt incidence (75%) over the control treatment, despite the challenge of inoculation with a highly pathogenic TOFU-IHBT strain. This was associated with significant expressions of the defense genes, indicating the induction of host resistance by a biocontrol consortium. In field experiments on two locations, the bioconsortium was highly effective in recording maximum mean fruit yields (54.5 and 60%) and a minimum mean vascular wilt incidence (37.5%) in comparison to the untreated control. Thus, Chaetomium-based bioconsortium demonstrated consistency in its performance across the two experiments in 2 years under the two field conditions.

Transcriptome Reprogramming of Tomato Orchestrate the Hormone Signaling Network of Systemic Resistance Induced by Chaetomium globosum.

Chaetomium globosum is a potential biological control agent effective against various plant pathogens. Several reports are available on the mycoparastism and antibiosis mechanisms of C. globosum against plant pathogenic fungi, whereas a few states induced resistance. The potential induced defense component of C. globosum (Cg-2) was evaluated against early blight disease of tomato (Solanum lycopersicum) and further, global RNA sequencing was performed to gain deep insight into its mechanism. The expression of marker genes of hormone signaling pathways, such as PR1, PiII, PS, PAL, Le4, and GluB were analyzed using real-time quantitative reverse transcription PCR (qRT-PCR) to determine the best time point for RNA sequencing. The transcriptome data revealed that 22,473 differentially expressed genes (DEGs) were expressed in tomato at 12 h post Cg-2 inoculation as compared with control plants and among these 922 DEGs had a fold change of -2 to +2 with p < 0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the DEGs were belonging to metabolic pathways, biosynthesis of secondary metabolites, plant-pathogen interaction, chlorophyll metabolism, and plant hormone signal transduction. Gene Ontology (GO) analysis revealed that DEGs were enriched mainly related to binding activity (GO:0005488), catalytic activity (GO:0003824), metabolic process (GO:0008152), cellular process (GO:0009987), response to stimulus (GO:0050896), biological regulation (GO:0065007), and transcription regulator activity (GO:0140110). The gene modulations in hormone signaling transduction, phenylpropanoid biosynthesis, and mitogen-activated protein kinases (MPK) signaling indicated the upregulation of genes in these pathways. The results revealed active participation of jasmonic acid (JA) and salicylic acid (SA) signaling transduction pathways which further indicated the involvement of induced systemic resistance (ISR) and systemic acquired resistance (SAR) in the systemic resistance induced by Cg-2 in tomato.

Identification and expression analysis of pathogenicity-related genes of Rhizoctonia solani anastomosis groups infecting rice.

Sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph; Thanatephorus cucumeris) is a major constraint in rice production. Among the different anastomosis groups (AGs) of Rhizoctonia solani, AG1-IA causes sheath blight of rice, which induce necrotic lesions on leaf sheaths of the infected plants. Several reports contradict the host specificity of anastomosis groups in Rhizoctonia solani. There is lack of information on the pathogenicity genes of these Rhizoctonia solani anastomosis groups during sheath blight infection in rice. In the present study, Rhizoctonia solani isolates collected from diverse rice growing regions of India were screened for anastomosis groups and two groups namely, AG1-IA, AG2-2 were identified. Accordingly, comparative studies were made with AG1-IA (GenBank ID: 16,395) and AG2-2 (GenBank ID: 2,318,768) group sequences, which enabled the identification of specific gene clusters (119 in AG1-IA and 604 in AG2-2) belonging to these groups. Pathogen Host Interaction (PHI) blast with these specific gene clusters could further identify genes involved in host pathogen interaction (38 in AG1_IA and 150 in AG2-2), which were shortlisted for qRT-PCR validation based on qcov cutoff values representing different phenotypic categories of PHI blast. Expression analysis-based validation in sheath blight susceptible (Pusa Basmati 1) and resistant (Pusa 1908-13-12-5) rice genotypes showed that most of the genes expressed significantly higher in the susceptible variety Pusa Basmati 1. The genes like inorganic phosphate transporter (AG1_IPT), Bromodomain containing protein (AG1_BrD), Aldehyde dehydrogenase (AG1_AldD), AMP binding domain (AG1_AMP) and Heme peroxidase (AG1_HmPr) were upregulated in the susceptible genotype, PB 1 at 72hpi compared to Pusa 1908-13-12-5. Among these, inorganic phosphate transporter (AG1_IPT), Bromodomain containing protein (AG1_BrD) and Heme peroxidase (AG1_HmPr) were specific to Rhizoctonia solani AG1-IA. Through the present study, we could demonstrate the AG1-IA-specific interactions of Rhizoctonia solani causing sheath blight disease of rice, which is a step forward in understanding the specificity of Rhizoctonia solani with reference to sheath blight disease of rice. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02934-1.

Overexpression of EcDREB2A transcription factor from finger millet in tobacco enhances tolerance to heat stress through ROS scavenging.

An extreme temperature regime beyond desired level imposes significant stress in crop plants. The low and high temperature stresses are one of the primary constraints for plant development and yield. Finger millet, being a climate resilient crop, is a potential source of novel stress tolerant genes. In this study, functional characterization of finger millet DREB2A gene in different abiotic stress conditions was done. This novel EcDREB2A transcription factor isolated from finger millet is a truncated version of DREB2A gene compared to previously reported DREB genes from other plant species. The overexpression of EcDREB2A in transgenic tobacco exhibits improved tolerance against heat stress 42 °C for up to 7 days, by altering physiology and biochemical means. However, same transgenic lines were unable to provide tolerance to 200 mM NaCl and 200 mM Mannitol stress. Under heat stress conditions, increased seed germination with improved lateral roots, fresh and dry weight relative to wild type (WT) was observed. The EcDREB2A transgenics exposed to heat stress showed improved rate of stomatal conductance, chlorophyll and carotenoids contents, and other photosynthesis parameters compared to WT plants. EcDREB2A overexpression also resulted in increased antioxidant enzyme activity (SOD, CAT, GR, POD and, APX) with decreased electrolyte leakage (EL), H2O2, and malondialdehyde (MDA) content than WT plants under heat stress. Quantitative real time expression analysis demonstrated that all eight downstream genes were significantly upregulated in transgenic plants only after heat stress. Our data provide a clear demonstration of the positive impact of overexpression of EcDREB2A providing heat stress tolerance to plants.

Allele mining for a drought responsive gene DRO1 determining root growth angle in donors of drought tolerance in rice (Oryza sativa L.).

Deeper Rooting 1 (DRO1) gene identified from a major QTL on chromosome 9 increases the root growth angle (RGA) and thus facilitates survival under drought and hence is an excellent candidate for rice improvement. Twenty-four major Indian upland and lowland genotypes including the 'yield under drought' (DTY) QTL donors were subjected to allele mining of DRO1 (3058 bp) using four pairs of overlapping primers. A total of 216 and 52 SNPs were identified across all genotypes in the gene and coding region (756 bp) respectively with transversions 3.6 fold more common than transitions in the gene and 2.5 times in the CDS. In 251 amino acid long protein, substitutions were found in 19 positions, wherein change in position 92 was the most frequent. Based on allele mining, the 24 genotypes can be classified into 16 primary structure variants ranging from complete functional allele (Satti, IR36 and DTY 3.1 donor, IR81896-B-B-195) to truncated non-functional alleles in PMK2, IR64, IR20 and Swarna. All the DTY donors, other than IR81896-B-B-195, and most of the upland drought tolerant cultivars (Nagina 22, Vandana and Dhagaddeshi) had accumulated 6-19 SNPs and 4-8 amino acid substitutions resulting in substantial differences in their protein structure. The expression analysis revealed that all the genotypes showed upregulation under drought stress though the degree of upregulation varied among genotypes. The information on structural variations in DRO1 gene will be very useful for the breeders, especially in the light of recent breeding programmes on improving drought tolerance using several DTY donors and upland accessions. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12298-021-00950-2).

Understanding Rice-Magnaporthe Oryzae Interaction in Resistant and Susceptible Cultivars of Rice under Panicle Blast Infection Using a Time-Course Transcriptome Analysis.

Rice blast is a global threat to food security with up to 50% yield losses. Panicle blast is a more severe form of rice blast and the response of rice plant to leaf and panicle blast is distinct in different genotypes. To understand the specific response of rice in panicle blast, transcriptome analysis of blast resistant cultivar Tetep, and susceptible cultivar HP2216 was carried out using RNA-Seq approach after 48, 72 and 96 h of infection with Magnaporthe oryzae along with mock inoculation. Transcriptome data analysis of infected panicle tissues revealed that 3553 genes differentially expressed in HP2216 and 2491 genes in Tetep, which must be the responsible factor behind the differential disease response. The defense responsive genes are involved mainly in defense pathways namely, hormonal regulation, synthesis of reactive oxygen species, secondary metabolites and cell wall modification. The common differentially expressed genes in both the cultivars were defense responsive transcription factors, NBS-LRR genes, kinases, pathogenesis related genes and peroxidases. In Tetep, cell wall strengthening pathway represented by PMR5, dirigent, tubulin, cell wall proteins, chitinases, and proteases was found to be specifically enriched. Additionally, many novel genes having DOMON, VWF, and PCaP1 domains which are specific to cell membrane were highly expressed only in Tetep post infection, suggesting their role in panicle blast resistance. Thus, our study shows that panicle blast resistance is a complex phenomenon contributed by early defense response through ROS production and detoxification, MAPK and LRR signaling, accumulation of antimicrobial compounds and secondary metabolites, and cell wall strengthening to prevent the entry and spread of the fungi. The present investigation provided valuable candidate genes that can unravel the mechanisms of panicle blast resistance and help in the rice blast breeding program.

Exploring the edible gum (galactomannan) biosynthesis and its regulation during pod developmental stages in clusterbean using comparative transcriptomic approach.

Galactomannan is a polymer of high economic importance and is extracted from the seed endosperm of clusterbean (C. tetragonoloba). In the present study, we worked to reveal the stage-specific galactomannan biosynthesis and its regulation in clusterbean. Combined electron microscopy and biochemical analysis revealed high protein and gum content in RGC-936, while high oil bodies and low gum content in M-83. A comparative transcriptome study was performed between RGC-936 (high gum) and M-83 (low gum) varieties at three developmental stages viz. 25, 39, and 50 days after flowering (DAF). Total 209,525, 375,595 and 255,401 unigenes were found at 25, 39 and 50 DAF respectively. Differentially expressed genes (DEGs) analysis indicated a total of 5147 shared unigenes between the two genotypes. Overall expression levels of transcripts at 39DAF were higher than 50DAF and 25DAF. Besides, 691 (RGC-936) and 188 (M-83) candidate unigenes that encode for enzymes involved in the biosynthesis of galactomannan were identified and analyzed, and 15 key enzyme genes were experimentally validated by quantitative Real-Time PCR. Transcription factor (TF) WRKY was observed to be co-expressed with key genes of galactomannan biosynthesis at 39DAF. We conclude that WRKY might be a potential biotechnological target (subject to functional validation) for developing high gum content varieties.