Unveiling the dynamic expression of PR-1 during Musa spp. infection by Fusarium oxysporum fsp. Cubense: a cloning and characterization study.

BACKGROUND: Pathogen-related proteins (PR) are pivotal in plant defense, combating diverse biotic and abiotic stresses. While multiple gene families contribute to banana resistance against Fusarium oxysporum f sp. cubense (Foc), Pseudocercospora eumusae, and Pratylenchus coffeae, the significance of PR-1 genes in defense is paramount. METHODS: Three PR-1 genes, up-regulated under diverse biotic stresses, were cloned from both resistant and susceptible cultivars of Foc, P. eumusae, and P. coffeae. Molecular characterization, phylogenetic analysis, and docking studies with the Foc TR4 CP gene were conducted. RESULTS: Through transcriptomic and real-time studies, three PR-1 genes (Ma02_g15050, Ma02_g15060, and Ma04_g34800) from Musa spp. were identified. These genes exhibited significant up-regulation in resistant cultivars when exposed to Foc, P. eumusae, and P. coffeae. Cloning of these genes was successfully performed from both resistant and susceptible cultivars of Foc race 1 and TR4, P. eumusae, and P. coffeae. Distinct characteristics were observed among the PR-1 genes, with groups 1 and 2 being acidic with signal peptides, and group 3 being basic without signal peptides. All cloned PR-1 proteins belonged to the CAP superfamily (PF00188). Phylogenetic analysis revealed clustering patterns for acidic PR-1 proteins, and KEGG orthology showed associations with vital pathways, including MAPK signaling, plant hormone signal transduction, and plant-pathogen interaction. Secondary and tertiary structure analyses confirmed sequence conservation across studied species. Docking studies explored interactions between the cerato-platanin (CP) gene from Foc TR4 and Ma02_g15060 from banana, suggesting the potential hindrance of PR-1 antifungal activity through direct interaction. CONCLUSIONS: The findings underscore the crucial role of cloned PR-1 genes in banana plant defense mechanisms against a broad spectrum of biotic stresses. These genes, especially those in groups 1 and 2, hold promise as candidates for developing stress-tolerant banana cultivars. The study provides valuable insights into the molecular aspects of banana defense strategies, emphasizing the potential applications of PR-1 genes in enhancing banana resilience.

MusaRgeneDB: an online comprehensive database for disease resistance genes in Musa spp.

Banana is one of the major food crops and its production is subject to many pests and diseases. Banana breeding exploits wild relatives and progenitor species for the introgression of resistant genes (R) into cultivated varieties to overcome these hurdles. With advances in sequencing technologies, whole-genome sequences are available for many Musa spp. and many of them are potential donors of disease resistance genes. Considering their potential role, R genes from these species were explored to develop an user-friendly open-access database that will be useful for studying and implementing disease resistance in bananas. MusaRgene database is complemented with complete details of 3598 R genes identified from eight Musa spp. and rice, Arabidopsis, sorghum along with its classification and separate modules on its expression under various stresses in resistant and susceptible cultivars and corresponding SSRs are also provided. This database can be regarded as the primary resource of information on R genes from bananas and their relatives. R genes from other allele mining studies are also incorporated which will enable the identification of its homolog in related Musa spp. MusaRgene database will aid in the identification of genes and markers associated, cloning of full-length R genes, and genetic transformation or gene editing of the R genes in susceptible cultivars. Multiple R genes can also be identified for pyramiding the genes to increase the level of resistance and durability. Overall, this database will facilitate the understanding of defense mechanisms in bananas against biotic or abiotic stresses leading to the development of promising disease-resistant varieties.

Genome-wide identification, characterization of expansin gene family of banana and their expression pattern under various stresses.

Expansin, a cell wall-modifying gene family, has been well characterized and its role in biotic and abiotic stress resistance has been proven in many monocots, but not yet studied in banana, a unique model crop. Banana is one of the staple food crops in developing countries and its production is highly influenced by various biotic and abiotic factors. Characterizing the expansin genes of the ancestor genome (M. acuminata and M. balbisiana) of present day cultivated banana will enlighten their role in growth and development, and stress responses. In the present study, 58 (MaEXPs) and 55 (MbaEXPs) putative expansin genes were identified in A and B genome, respectively, and were grouped in four subfamilies based on phylogenetic analysis. Gene structure and its duplications revealed that EXPA genes are highly conserved and are under negative selection whereas the presence of more number of introns in other subfamilies revealed that they are diversifying. Expression profiling of expansin genes showed a distinct expression pattern for biotic and abiotic stress conditions. This study revealed that among the expansin subfamilies, EXPAs contributed significantly towards stress-resistant mechanism. The differential expression of MaEXPA18 and MaEXPA26 under drought stress conditions in the contrasting cultivar suggested their role in drought-tolerant mechanism. Most of the MaEXPA genes are differentially expressed in the root lesion nematode contrasting cultivars which speculated that this expansin subfamily might be the susceptible factor. The downregulation of MaEXPLA6 in resistant cultivar during Sigatoka leaf spot infection suggested that by suppressing this gene, resistance may be enhanced in susceptible cultivar. Further, in-depth studies of these genes will lead to gain insight into their role in various stress conditions in banana. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-021-03106-x.

Development of fusarium wilt resistant mutants of Musa spp. cv.Rasthali (AAB, Silk subgroup) and comparative proteomic analysis along with its wild type.

MAIN CONCLUSION: Induced mutagenesis using embryogenic cell suspension (ECS) explants with toxin based screening is an effective tool to create non-chimeral Fusarium wilt resistant mutants in banana. Global proteomics unravel the molecular mechanism behind resistance. Race 1 of Fusarium wilt is a serious threat to Musa spp. cv.Rasthali (AAB, Silk subgroup) which is a choice variety traditionally grown in most of the south East Asian countries. Resistant gene introgression into susceptible varieties through conventional breeding has several limitations and the predominant ones being sterility and long generation time. Under such circumstances, induced mutagenesis combined with toxin based in vitro screening remains as the viable alternative for the development of fusarium wilt resistant Rasthali. Therefore, induced mutagenesis was attempted by using ethylmethane sulfonate (EMS) in embryogenic cell suspension (ECS) of Rasthali followed by in vitro screening for fusarium wilt resistance using new generation toxins and pot screening through challenge inoculation with Foc race 1. This ultimately resulted in the identification of 15 resistant lines. Global proteomic analysis in one of the resistant mutant lines namely NRCBRM15 and its wild type revealed 37 proteins, of which 20 showed differential expression. Out of 20 proteins, nineteen were significantly abundant in NRCBRM15 and only one was abundant in wild Rasthali. A total of nine genes based on protein expression were further validated using quantitative real time polymerase chain reaction (qRT-PCR). Annotation results revealed that some of the genes namely Enolase, ATP synthase-alpha subunit, Actin 2, Actin 3,-glucanase, UTP-glucose-1-phosphate uridylyltransferase, Respiratory burst oxidase homolog, V type proton ATPase catalytic subunit A and DUF292 domain containing protein are involved in diverse functions such as carbohydrate metabolism, energy production, electron carrier, response to wounding, binding proteins, cytoskeleton organization, extracellular region, structural molecule and defense.

Genome-wide analysis of pathogenesis-related protein 1 (PR-1) gene family from Musa spp. and its role in defense response during stresses.

Pathogenesis related protein-1 (PR-1) is the most abundantly produced protein during defense response against many biotic and abiotic stresses. However, knowledge on PR-1 gene family and its evolutionary relationship in banana is very limited. In order to study the potential role of PR-1 genes in banana, genome wide identification, structure analysis and expressions were performed. A total of 15 and 11 PR-1 genes were identified from A and B genomes of banana and the proteins encoded by this gene family are of varying lengths and harbor conserved domains and motifs. PR-1 genes are unevenly dispersed on 11 chromosomes with segmental duplication in both A and B genome, suggesting an important contribution of duplication in expansion of PR-1 gene family in banana. qRT-PCR analysis of PR-1 gene showed positive correlation with the RNAseq data under various stresses and examination of expression pattern of selected MaPR-1 genes in banana revealed its role in biotic and abiotic stresses in general and fusarium wilt in particular. This study provides significant insight into the functions of PR-1 genes which can be further exploited as a promising candidate for developing multiple stress tolerant banana varieties.

A novel temporary immersion bioreactor system for large scale multiplication of banana (Rasthali AAB-Silk).

Musa sp. cultivar Rasthali (Silk AAB) is a choice variety of the Asian sub-continent. Its production and sustenance are threatened by Fusarium wilt, which affects the livelihoods of small and marginal farmers. The use of quality planting material is one of the strategies to manage the disease. Availability of quality planting material for varieties other than Grand Naine is limited. Large-scale micropropagation using existing technologies is laborious and expensive. Temporary immersion bioreactor system is emerging as a potential advancement in the micropropagation industry. In this study, a cost-effective temporary immersion bioreactor (TIB) system has been developed and an efficient micropropagation method has been standardized. Explants cultured in TIB with 250 ml of culture medium in a 2-min immersion frequency of 6 h were found to be efficient for shoot proliferation and rooting. Its efficacy has been compared with the semisolid culture method. At the end of the 6th subculture, 1496 ± 110 shoots per explant were obtained in TIB. Chlorophyll, carotenoid, stomatal index, and the number of closed stomata were examined to determine the physiological functions of the plants grown in TIB and compared with semisolid grown plantlets. Plantlets grown in TIB were genetically stable and were confirmed using inter-simple sequence repeat (ISSR) markers. The multiplication of shoots in TIB was 2.7-fold higher than the semisolid culture method, which is suitable for large-scale production of planting material for commercial applications.

Decoding the molecular mechanism of parthenocarpy in Musa spp. through protein-protein interaction network.

Banana, one of the most important staple fruit among global consumers is highly sterile owing to natural parthenocarpy. Identification of genetic factors responsible for parthenocarpy would facilitate the conventional breeders to improve the seeded accessions. We have constructed Protein-protein interaction (PPI) network through mining differentially expressed genes and the genes used for transgenic studies with respect to parthenocarpy. Based on the topological and pathway enrichment analysis of proteins in PPI network, 12 candidate genes were shortlisted. By further validating these candidate genes in seeded and seedless accession of Musa spp. we put forward MaAGL8, MaMADS16, MaGH3.8, MaMADS29, MaRGA1, MaEXPA1, MaGID1C, MaHK2 and MaBAM1 as possible target genes in the study of natural parthenocarpy. In contrary, expression profile of MaACLB-2 and MaZEP is anticipated to highlight the difference in artificially induced and natural parthenocarpy. By exploring the PPI of validated genes from the network, we postulated a putative pathway that bring insights into the significance of cytokinin mediated CLAVATA(CLV)-WUSHEL(WUS) signaling pathway in addition to gibberellin mediated auxin signaling in parthenocarpy. Our analysis is the first attempt to identify candidate genes and to hypothesize a putative mechanism that bridges the gaps in understanding natural parthenocarpy through PPI network.

Proteomic analysis of somatic embryo development in Musa spp. cv. Grand Naine (AAA).

Somatic embryos are comparable to their zygotic counterparts for morphological traits but are derived from somatic cells through various metabolic regulations, collectively referred as somatic embryogenesis (SE). It has been well exploited for germplasm conservation, genetic engineering, mutation breeding, for artificial seed technology and as a tool for mass multiplication. Though somatic embryo development is an important area of interest in growth, and developmental studies, the underlying molecular mechanism remains unclear. Therefore, understanding the molecular basis behind somatic embryo development can provide insight into the signaling pathways integrating this process. Proteomic analysis of somatic embryo development in cv. Grand Naine (AAA) was carried out to identify the differentially expressed protein during somatic embryo development stages, using two dimensional gel electrophoresis together with mass spectrometry. In total, 25 protein spots were differentially expressed during sequential developmental stages of somatic embryos. Among these, three proteins were uniquely present in 30 days globular stage and six proteins in 60 days old mature somatic embryo. Functional annotation of identified spots showed that major proteins are involved in growth and developmental process (17%) followed by defense response (12%) and signal transportation events (12%). In the early stage, cell division and growth related proteins are involved in the induction of somatic embryos whereas in the late developmental stage, cell wall associated proteins along with stress related proteins played a defensive role against dehydration and osmotic stress and resulted in the maturation of somatic embryo. The identified stage specific proteins are valuable indicators and genetic markers for screening and for media manipulation to improve SE efficiency in recalcitrant crops and varieties.

MusatransSSRDB (a transcriptome derived SSR database) - An advanced tool for banana improvement.

Availability of transcriptome datasets for use in accelerated molecular-based breeding in Musa species is limited. Illumina Hiseq technology was employed to determine differential gene expression between the contrasting cultivars for three different stresses (Eumusae leaf spot -Mycosphaerella eumusae, root lesion nematode - Pratylenchus coffeae and moisture deficit stress) under challenged and unchallenged conditions. An average of 34.72 million of reads was assembled into ~47629 contigs, and ~5,466 simple sequence repeats (SSR) from each library were identified. GO annotation and KEGG pathway analysis were carried for all the transcripts and the SSR, SNPs were also detected. Based on this information, a MusatransSSRDB has been developed. Currently, the database consists of 32,800 SSRs with the unique information like putative function of the SSR-containing genes and their metabolic pathway and expression profiling under various stress conditions. This database provides information on in silico polymorphic SSRs (2830 SSRs) between the contrasting cultivars for each stress and within stress. Information on in silico polymorphic SSRs specific to differentially expressed genes under challenged condition for each stress can also be accessed. This database facilitates the retrieval of results by navigating the tabs for cultivars, stress and polymorphism. This database was developed using HTML, Java and PHP; datasets are stored in MySQL database and accessible in the public domain (http://bioinfnrcb.byethost7.com/nrcbbio/). This unique information facilitates the banana breeder to select the SSR primers based on specific objectives. MusatransSSRDB along with other genomics databases will facilitate the genetic dissection and breeding for complex traits in banana. Thus, this database is a step forward in economizing cost, time, manpower and other resources. Keywords.

Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine.

KEY MESSAGE: Endogenous hormone secretion proteins along with stress and defense proteins play predominant role in banana embryogenesis. This study reveals the underlying molecular mechanism during transition from vegetative to embryogenic state. Banana (Musa spp.) is well known globally as a food fruit crop for millions. The requirement of quality planting material of banana is enormous. Although mass multiplication through tissue culture is in vogue, high-throughput techniques like somatic embryogenesis (SE) as a mass multiplication tool needs to be improved. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. SE in banana is completely genome-dependent and most of the commercial cultivars exhibit recalcitrance. Thus, understanding the molecular basis of embryogenesis in Musa will help to develop strategies for mass production of quality planting material. In this study, differentially expressed proteins between embryogenic calli (EC) and non-embryogenic calli (NEC) with respect to the explant, immature male flower buds (IMFB), of cv. Grand Naine (AAA) were determined using two-dimensional gel electrophoresis (2DE). The 2DE results were validated through qRT-PCR. In total, 65 proteins were identified: 42 were highly expressed and 23 were less expressed in EC compared to NEC and IMFB. qRT-PCR analysis of five candidate proteins, upregulated in EC, were well correlated with expression at transcript level. Further analysis of proteins showed that embryogenesis in banana is associated with the control of oxidative stress. The regulation of ROS scavenging system and protection of protein structure occurred in the presence of heat shock proteins. Alongside, high accumulation of stress-related cationic peroxidase and plant growth hormone-related proteins like indole-3-pyruvate monooxygenase and adenylate isopentenyltransferase in EC revealed the association with the induction of SE.

Transcriptomic Changes of Drought-Tolerant and Sensitive Banana Cultivars Exposed to Drought Stress.

In banana, drought responsive gene expression profiles of drought-tolerant and sensitive genotypes remain largely unexplored. In this research, the transcriptome of drought-tolerant banana cultivar (Saba, ABB genome) and sensitive cultivar (Grand Naine, AAA genome) was monitored using mRNA-Seq under control and drought stress condition. A total of 162.36 million reads from tolerant and 126.58 million reads from sensitive libraries were produced and mapped onto the Musa acuminata genome sequence and assembled into 23,096 and 23,079 unigenes. Differential gene expression between two conditions (control and drought) showed that at least 2268 and 2963 statistically significant, functionally known, non-redundant differentially expressed genes (DEGs) from tolerant and sensitive libraries. Drought has up-regulated 991 and 1378 DEGs and down-regulated 1104 and 1585 DEGs respectively in tolerant and sensitive libraries. Among DEGs, 15.9% are coding for transcription factors (TFs) comprising 46 families and 9.5% of DEGs are constituted by protein kinases from 82 families. Most enriched DEGs are mainly involved in protein modifications, lipid metabolism, alkaloid biosynthesis, carbohydrate degradation, glycan metabolism, and biosynthesis of amino acid, cofactor, nucleotide-sugar, hormone, terpenoids and other secondary metabolites. Several, specific genotype-dependent gene expression pattern was observed for drought stress in both cultivars. A subset of 9 DEGs was confirmed using quantitative reverse transcription-PCR. These results will provide necessary information for developing drought-resilient banana plants.