Sequencing and Characterization of M. morganii Strain UM869: A Comprehensive Comparative Genomic Analysis of Virulence, Antibiotic Resistance, and Functional Pathways.

Morganella morganii is a Gram-negative opportunistic Enterobacteriaceae pathogen inherently resistant to colistin. This species causes various clinical and community-acquired infections. This study investigated the virulence factors, resistance mechanisms, functional pathways, and comparative genomic analysis of M. morganii strain UM869 with 79 publicly available genomes. The multidrug resistance strain UM869 harbored 65 genes associated with 30 virulence factors, including efflux pump, hemolysin, urease, adherence, toxin, and endotoxin. Additionally, this strain contained 11 genes related to target alteration, antibiotic inactivation, and efflux resistance mechanisms. Further, the comparative genomic study revealed a high genetic relatedness (98.37%) among the genomes, possibly due to the dissemination of genes between adjoining countries. The core proteome of 79 genomes contains the 2692 core, including 2447 single-copy orthologues. Among them, six were associated with resistance to major antibiotic classes manifested through antibiotic target alteration (PBP3, gyrB) and antibiotic efflux (kpnH, rsmA, qacG; rsmA; CRP). Similarly, 47 core orthologues were annotated to 27 virulence factors. Moreover, mostly core orthologues were mapped to transporters (n = 576), two-component systems (n = 148), transcription factors (n = 117), ribosomes (n = 114), and quorum sensing (n = 77). The presence of diversity in serotypes (type 2, 3, 6, 8, and 11) and variation in gene content adds to the pathogenicity, making them more difficult to treat. This study highlights the genetic similarity among the genomes of M. morganii and their restricted emergence, mostly in Asian countries, in addition to their growing pathogenicity and resistance. However, steps must be taken to undertake large-scale molecular surveillance and to direct suitable therapeutic interventions.

Development of SNP markers linked to purple blotch resistance for marker-assisted selection in onion (Allium cepa L.) breeding.

Purple blotch (PB), caused by Alternaria porri (Ellis) Cifferi, is one of the most destructive diseases of onion worldwide. Rapid development and deployment of resistant onion varieties is the most effective approach to control this disease. A single dominant gene, ApR1 was previously linked to PB resistance in onion cultivar 'Arka Kalyan'. In this study, an advanced RIL population derived from a cross between the resistant (Arka Kalyan) and susceptible (Agrifound Rose) cultivar of onion was used to fine map the resistant locus with SNP markers. Twenty plants from the RIL population, ten each with disease resistance and susceptibility trait, were subjected to restriction site-associated DNA sequencing (RAD-Seq) and generated 7388 single nucleotide polymorphisms (SNPs). Correlation analysis between marker genotypes and PB disease phenotype on the 20 plants identified 27 SNPs as candidate markers linked to ApR1 gene for PB resistance. Six candidate SNPs were converted to Kompetitive Allele-Specific PCR (KASP) markers designated as ApRsnip5, ApRsnip8, ApRsnip14, ApRsnip21, ApRsnip23 and ApRsnip25. Marker-trait association based on disease phenotyping and KASP genotyping data on 153 RILs confirmed that all six KASP markers were tightly associated with ApR1 gene within the genetic distance of 1.3 CentiMorgan (cM). ApRsnip14 co-segregated with the ApR1 locus. Further, the six KASP markers were tested on 27 onion lines with different genetic backgrounds. ApRsnip14, ApRsnip21, ApRsnip5 and ApRsnip23 not only showed the correct resistance allele in 3 resistance genotypes, but also clustered together in the remaining 24 susceptible lines. Alternatively, ApRsnip8 and ApRsnip25 exhibited false positives in two onion lines which do not have the R-gene. Overall, our results suggest that ApRsnip14 and ApRsnip23 with their close linkage to ApR1 locus and greater applicability on breeding germplasm are recommended in marker-assisted selection for PB resistance in onion breeding program. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03562-7.

Cross-kingdom small RNA communication between plants and fungal phytopathogens-recent updates and prospects for future agriculture.

Small RNAs (sRNAs) are short non-coding regulatory RNA sequences that silence the complementary expressive transcripts through an endogenous RNA mediated interference mechanism (RNAi). These sRNAs typically move through plasmodesmata and phloem in plants to support disease resistance, and also through septal pores and vesicles in fungi to act as effector of pathogenicity. Notably, recent reports have shown the occurrence of a bidirectional trafficking of these sRNAs between the host plants and the attacking fungal phytopathogen which have significant implication in the nature of the infection. While the trans-species sRNAs from the pathogen can silence the host mRNAs and inhibit the host immunity genes, the sRNA modules from the host plants can silence the mRNA in the pathogen by impeding the expression of the pathogenicity-related genes. In the present review, we discuss the current state of sRNA trafficking between the plant and the pathogen with special emphasis on the mechanism of cross-kingdom communication which could contribute to the development of pathogen and pest control in future agriculture.

Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.

A single transcript CRISPR/Cas9 mediated mutagenesis of CaERF28 confers anthracnose resistance in chilli pepper (Capsicum annuum L.).

MAIN CONCLUSION: T-DNA-free homozygous mutant lines developed through a single transcript CRISPR/Cas9 system harboring the desired modification in the CaERF28 locus exhibited significantly enhanced resistance to the anthracnose pathogen Colletotrichum truncatum coupled with the improved expression of defense responsive genes. Anthracnose, caused by Colletotrichum species, is a major disease of chilli (Capsicum annuum) accounting for significant pre- and post-harvest yield losses across the tropical and subtropical regions of the world. Management of chilli anthracnose using traditional methods have not met with noticeable success. In the present study, we have demonstrated an enhanced anthracnose resistance through a single transcript unit CRISPR/Cas9 mediated alteration of the susceptibility gene CaERF28 in C. annuum. A construct with a single Pol II promoter-driven expression of Cas9, sgRNA and a hammerhead ribozyme (RZ) was designed to modify the CaERF28 gene in the susceptible chilli genotype Arka Lohit. Fourty-five C-ERF28-induced mutant lines (72.5%) were identified from 62 T0 transgenic plants. Further, simultaneously targeted multiple sites within CaERF28 showed increased mutation (85.7%) efficiency. DNA sequence analysis showed that these plants harboured multiple InDels at the target site. The allelic mutants of C-ERF28 were transferred to the following generations by simple Mendelian inheritance. Segregation in the T1 and T2 generations resulted in the identification of T-DNA free and marker-free C-ERF28 mutant lines. Five homozygous mutants demonstrated enhanced resistance to anthracnose compared to wild type as demonstrated by reduced spore count and fungal growth as well as induced expression of defense-related genes. Our results demonstrated that the STU-CRISPR/Cas9 mediated editing of the CaERF28 gene is a rapid, safe and versatile approach for enhancing anthracnose resistance in chilli pepper and pave way for its utilization in the improvement of other solanaceous crops.

Genome wide identification and expression analysis of pepper C2H2 zinc finger transcription factors in response to anthracnose pathogen Colletotrichum truncatum.

Although, the C2H2 zinc finger (ZF) family of plant transcription factors have been implicated in multiple biological processes, they are yet to be characterized in the economically important chilli pepper (Capsicum annuum). In this study, a total of 79 C2H2 ZF genes were identified in the pepper genome. Phylogenetic analysis categorized the pepper C2H2 ZF (CaZF) members into five subfamilies each with unique conserved domains and functions. Genomic organization revealed that CaZF genes have variable number of introns consistent with the characteristics defined by the evolutionary analysis. Segmental duplication-based purifying selection contributed to the expansion of CaZF genes in pepper. Additionally, 11 CaZF genes were identified as targets for 38 miRNAs indicating their role in post-transcriptional silencing-mediated genetic regulation. Gene expression analysis revealed that 18 CaZF genes were differentially expressed post-infection with the anthrocnose pathogen Colletotrichum truncatum, uncovering their potential function in pepper response to biotic stresses. Moreover, CaZFs were significantly induced post-treatment with methyl jasmonate and ethylene indicating their role in defense signaling. Notably, the MeJA responsive cis-elements were detected in the promoter regions of majority of CaZF genes, suggesting that CaZFs may be implicated in defense-responsive signal cross talking. Additionally, 18 CaZF genes were differentially expressed under drought and heat treatment, indicating their involvement in plant response to abiotic stresses. Overall, a comprehensive analysis of CaZF gene family in pepper provided significant insights into the understanding of C2H2 ZF-mediated stress regulation network, which would benefit the genetic improvement of pepper and other allied plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02601-x.

Engineering drought tolerance in plants through CRISPR/Cas genome editing.

Drought stress is primarily responsible for heavy yield losses and productivity in major crops and possesses the greatest threat to the global food security. While conventional and molecular breeding approaches along with genetic engineering techniques have been instrumental in developing drought-tolerant crop varieties, these methods are cumbersome, time consuming and the genetically modified varieties are not widely accepted due to regulatory concerns. Plant breeders are now increasingly centring towards the recently available genome-editing tools for improvement of agriculturally important traits. The advent of multiple sequence-specific nucleases has facilitated precise gene modification towards development of novel climate ready crop variants. Amongst the available genome-editing platforms, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) system has emerged as a revolutionary tool for its simplicity, adaptability, flexibility and wide applicability. In this review, we focus on understanding the molecular mechanism of drought response in plants and the application of CRISPR/Cas genome-editing system towards improved tolerance to drought stress.

Base editing in crops: current advances, limitations and future implications.

Targeted mutagenesis via genome-editing technologies holds great promise in developing improved crop varieties to meet future demands. Point mutations or single nucleotide polymorphisms often determine important agronomic traits of crops. Genome-editing-based single-base changes could generate elite trait variants in crop plants which help in accelerating crop improvement. Among the genome-editing technologies, base editing has emerged as a novel and efficient genome-editing approach which enables direct and irreversible conversion of one target base into another in a programmable manner. A base editor is a fusion of catalytically inactive CRISPR-Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations in the target region enabling precise editing of genomes. In the present review, we have summarized the development of different base-editing platforms. Then, we have focussed on the current advances and the potential applications of this precise technology in crop improvement. The review also sheds light on the limitations associated with this technology. Finally, the future perspectives of this emerging technology towards crop improvement have been highlighted.

Transcriptome analysis of a rice cultivar reveals the differentially expressed genes in response to wild and mutant strains of Xanthomonas oryzae pv. oryzae.

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating disease in most of the rice growing regions worldwide. Among the 42 BB resistance (R) genes, Xa23 is an executor R gene, conferring broad-spectrum disease resistance to all naturally occurring biotypes of Xoo. In this study, CBB23, a rice line carrying Xa23 gene, was inoculated with wild PXO99A and its mutant, P99M2, to retrieve the differentially expressed genes (DEGs). RNA-Seq analysis retrieved 1,235 DEGs (p-value ≤ 0.05) at 12, 24, 36, and 48 hours of post inoculation (hpi). Gene ontology (GO) analysis classified the DEGs functionally into biological process, cellular component and molecular function. KEGG pathway analysis categorized the DEGs into 11 different pathways, and the ribosome is a prominent pathway followed by biosynthesis of phenylpropanoids. Gene co-expression network analysis identified the clusters of transcription factors (TFs) which may be involved in PXO99A resistance. Additionally, we retrieved 67 differentially expressed TFs and 26 peroxidase responsive genes which may be involved in disease resistance mechanism. DEGs involved in the host-pathogen interaction, e.g., signaling mechanism, cell wall and plant hormones were identified. This data would be a valuable resource for researchers to identify the candidate genes associated with Xoo resistance.

Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.).

Anthracnose, caused by Colletotrichum spp. is the most devastating disease of chili (Capsicum annuum) in the tropical and subtropical regions of the world. The present study aimed at molecular mapping and development of markers linked to a new gene for anthracnose resistance in the chili cultivar 'Punjab Lal'. Phenotypic evaluation of F1, F2, and BC1F1 populations derived from a cross between 'Punjab Lal' and susceptible cultivar 'Arka Lohit' against a virulent isolate of C. truncatum revealed that anthracnose resistance in Punjab Lal is governed by a monogenic-dominant gene designated as RCt1. Forty-four (28 ISSRs and 16 AFLPs) out of 201 markers exhibited parental polymorphism and were used in bulk segregant analysis. Three ISSRs (ISSR411493, ISSR581485, and ISSR1121857) and one AFLP marker (E-ACA/M-CTG516) showed precise polymorphism between resistant and susceptible bulks, and were used for genotyping F2 and BC1 populations. The four putative fragments were converted into sequence-tagged site (STS) markers and southern blotting confirmed their association with the resistance locus. Molecular mapping revealed that the STS markers CtR-431 and CtR-594 were closely linked to the RCt1 locus in coupling at distances of 1.8 and 2.3 cM, respectively. Furthermore, both of these markers showed the presence of resistance-linked allele in seven genotypes including the highly resistant C. chinnese 'PBC932' and C. baccatum 'PBC80' while negatively validated in 32 susceptible genotypes. Therefore, CtR431 and CtR-594 could be recommended as efficient diagnostic markers to facilitate the introgression of RCt1 locus into susceptible chili variants towards the development of high-yielding anthracnose resistance genotypes in C. annuum background.

Analysis of microRNAs and their targets from onion (Allium cepa) using genome survey sequences (GSS) and expressed sequence tags (ESTs).

MicroRNAs are small non-coding RNAs of 21-24 nucleotides in length that acts as important modulators of gene expression related to numerous biological processes including development and defense response in eukaryotes. However, only a limited report on onion (Allium cepa) miRNAs is available and their associated role in growth and development of onion is not yet clear. Therefore, it is of interest to identify miRNAs and their targets in Allium cepa using the genome survey sequences (GSSs) and expressed sequence tags (ESTs) and deduce the functions of the target genes using gene ontology (GO) terms. We report 14 potential miRNAs belonging to 13 different families (miR162, miR168, miR172c, miR172e, miR398, miR400, miR414, miR1134, miR1223, miR6219, miR7725, miR8570, miR8703 and miR8752). BLAST analysis using psRNATarget server predicted 39 potential targets for the identified miRNAs majority of which were transcription factors implicated in plant growth, development, hormone signaling and stress responses. These data forms the basis for further analysis and verification towards understanding the miRNA mediated regulatory mechanism in Allium cepa.

Genome Editing in Rice: Recent Advances, Challenges, and Future Implications.

Rice (Oryza sativa L.) is the major food source for more than three billion people of the world. In the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties. However, stagnation in rice yield has been reported in recent decade owing to several factors including the emergence of pests and phyto pathogens, climate change, and other environmental issues posing great threat to global food security. There is an urgent need to produce more rice and associated cereals to satisfy the mammoth task of feeding a still growing population expected to reach 9.7 billion by 2050. Advances in genomics and emergence of multiple genome-editing technologies through use of engineered site-specific nucleases (SSNs) have revolutionized the field of plant science and agriculture. Among them, the CRISPR/Cas9 system is the most advanced and widely accepted because of its simplicity, robustness, and high efficiency. The availability of huge genomic resources together with a small genome size makes rice more suitable and feasible for genetic manipulation. As such, rice has been increasingly used to test the efficiency of different types of genome editing technologies to study the functions of various genes and demonstrate their potential in genetic improvement. Recently developed approaches including CRISPR/Cpf1 system and base editors have evolved as more efficient and accurate genome editing tools which might accelerate the pace of crop improvement. In the present review, we focus on the genome editing strategies for rice improvement, thereby highlighting the applications and advancements of CRISPR/Cas9 system. This review also sheds light on the role of CRISPR/Cpf1 and base editors in the field of genome editing highlighting major challenges and future implications of these tools in rice improvement.

Can-miRn37a mediated suppression of ethylene response factors enhances the resistance of chilli against anthracnose pathogen Colletotrichum truncatum L.

Pepper anthracnose, caused by Colletotrichum species complex is the most destructive disease of chilli (Capsicum annuum L.). miRNAs are key modulators of transcriptional and post- transcriptional expression of genes during defense responses. In the present study, we performed a comparative miRNA profiling of susceptible (Arka Lohit-AL) and resistant (Punjab Lal-PL) chilli cultivars to identify 35 differentially expressed miRNAs that could be classified as positive, negative or basal regulators of defense against C. truncatum, the most potent anthracnose pathogen. Interestingly, a novel microRNA can-miRn37a was significantly induced in PL but largely repressed in AL genotype post pathogen attack. Subsequent over-expression of can-miRn37a in AL showed enhanced resistance to anthracnose, as evidenced by decreased fungal growth and induced expression of defense-related genes. Consequently, the expression of its three target genes encoding the ethylene response factors (ERFs) was down-regulated in PL as well as in the over-expression lines of AL genotypes. The ability of these targets to be regulated by can-miRn37a was further confirmed by transient co-expression in Nicotiana benthamiana. Additionally, the virus-induced silencing of the three targets in the susceptible AL cultivar revealed their role in fungal colonization and induction of C. truncatum pathogenicity in chilli. Taken together, our study suggests that can-miRn37a provides a potential miRNA mediated approach of engineering anthracnose resistance in chilli by repressing ERFs and preventing fungal colonization.

Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. Cepae.

The basal plate rot fungus, Fusarium oxysporum f. sp. cepae (FOC), is the most devastating pathogen posing a serious threat to garlic (Allium sativum L.) production worldwide. MicroRNAs (miRNAs) are key modulators of gene expression related to development and defense responses in eukaryotes. However, the miRNA species associated with garlic immunity against FOC are yet to be explored. In the present study, a small RNA library developed from FOC infected resistant garlic line was sequenced to identify immune responsive miRNAs. Forty-five miRNAs representing 39 conserved and six novel sequences responsive to FOC were detected. qRT-PCR analyses further classified them into three classes based on their expression patterns in susceptible line CBT-As11 and in the resistant line CBT-As153. North-blot analyses of six selective miRNAs confirmed the qRT-PCR results. Expression studies on a selective set of target genes revealed a negative correlation with the complementary miRNAs. Furthermore, transgenic garlic plant overexpresing miR164a, miR168a and miR393 showed enhanced resistance to FOC, as revealed by decreased fungal growth and up-regulated expression of defense-responsive genes. These results indicate that multiple miRNAs are involved in garlic immunity against FOC and that the overexpression of miR164a, miR168a and miR393 can augment garlic resistance to Fusarium basal rot infection.