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Fusarium wilt of banana is a destructive widespread disease caused by Fusarium oxysporum f. sp. cubense (Foc) that ravaged banana plantations globally, incurring huge economic losses. Current knowledge demonstrates the involvement of several transcription factors, effector proteins, and small RNAs in the Foc-banana interaction. However, the precise mode of communication at the interface remains elusive. Cutting-edge research has emphasized the significance of extracellular vesicles (EVs) in trafficking the virulent factors modulating the host physiology and defence system. EVs are ubiquitous inter- and intra-cellular communicators across kingdoms. This study focuses on the isolation and characterization of Foc EVs from methods that make use of sodium acetate, polyethylene glycol, ethyl acetate, and high-speed centrifugation. Isolated EVs were microscopically visualized using Nile red staining. Further, the EVs were characterized using transmission electron microscopy, which revealed the presence of spherical, double-membrane, vesicular structures ranging in size from 50 to 200 nm (diameter). The size was also determined using the principle based on Dynamic Light Scattering. The Foc EVs contained proteins that were separated using SDS-PAGE and ranged between 10 and 315 kDa. Mass spectrometry analysis revealed the presence of EV-specific marker proteins, toxic peptides, and effectors. The Foc EVs were found to be cytotoxic, whose toxicity increased with EVs isolated from the co-culture preparation. Taken together, a better understanding of Foc EVs and their cargo will aid in deciphering the molecular crosstalk between banana and Foc.

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Cyanobacteria are a large group of ubiquitously found photosynthetic prokaryotes that are constantly exposed to different kinds of stressors of varying intensities and seem to overcome these in a precise and regulated manner. However, a high dose and duration of given stress induce cell death in a few select cyanobacteria, mainly to protect other cells (altruism). Despite the recent findings for the presence of biochemical and molecular hallmarks of cell death in cyanobacteria, it is yet a sketchily understood phenomenon. Regulation of metacaspase-like genes during Programmed Cell Death suggests it to be a genetically controlled mechanism like other eukaryotes. In addition to providing a comprehensive understanding of the current status of cell death in cyanobacteria, this review has used in silico analyses to directly compare the existence of some important molecular players operating in the intrinsic and extrinsic apoptotic pathways. Phylogenetic trees for all sequences indicate a cluster with a common ancestry and also a divergence from sequences of eukaryotic origin. To the best of our knowledge, such a comparison (except for orthocaspases) has not been attempted earlier and hopes to encourage workers in the field to investigate this altruistic phenomenon in detail.

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Biotic and abiotic stress factors drastically limit plant growth and development as well as alter the physiological, biochemical and cellular processes. This negatively impacts plant productivity, ultimately leading to agricultural and economical loss. Plant defense mechanisms elicited in response to these stressors are crucially regulated by the intricate crosstalk between defense hormones such as jasmonic acid (JA), salicylic acid and ethylene. These hormones orchestrate adaptive responses by modulating the gene regulatory networks leading to sequential changes in the root architecture, cell wall composition, secondary metabolite production and expression of defense-related genes. Fusarium wilt is a widespread vascular disease in plants caused by the soil-borne ascomycete Fusarium oxysporum and is known to attack several economically important plant cultivars. JA along with its conjugated forms methyl jasmonate and jasmonic acid isoleucine critically tunes plant defense mechanisms by regulating the expression of JA-associated genes imparting resistance phenotype. However, it should be noted that some members of F. oxysporum utilize the JA signaling pathway for disease development leading to susceptibility in plants. Therefore, JA signaling pathway becomes one of the important targets amenable for modulation to develop resistance response against Fusarium wilt in plants. In this review, we have emphasized on the physiological and molecular aspects of JA and its significant role in mounting an early defense response against Fusarium wilt disease. Further, utilization of the inherent JA signaling pathway and/or exogenous application of JA in generating Fusarium wilt resistant plants is discussed.

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Programmed cell death (PCD) is a genetically controlled process for the selective removal of damaged cells. Though understanding about plant PCD has improved over years, the mechanisms are yet to be fully deciphered. Among the several molecular players of PCD in plants, B cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family of co-chaperones are evolutionary conserved and regulate cell death, growth and development. In this study, we performed a genome-wide in silico analysis of the MusaBAG gene family in a globally important fruit crop banana. Thirteen MusaBAG genes were identified, out of which MusaBAG1, 7 and 8 genes were found to have multiple copies. MusaBAG genes were distributed on seven out of 11 chromosomes in banana. Except for one paralog of MusaBAG8 all the other 12 proteins have characteristic BAG domain. MusaBAG1, 2 and 4 have an additional ubiquitin-like domain whereas MusaBAG5-8 have a calmodulin binding motif. Most of the MusaBAG proteins were predicted to be localized in the nucleus and mitochondria or chloroplast. The in silico cis-regulatory element analysis suggested regulation associated with photoperiodic control, abiotic and biotic stress. The phylogenetic analysis revealed 2 major clusters. Digital gene expression analysis and quantitative real-time RT-PCR depicted the differential expression pattern of MusaBAG genes under abiotic and biotic stress conditions. Further studies are warranted to uncover the role of each of these proteins in growth, PCD and stress responses so as to explore them as candidate genes for engineering transgenic banana plants with improved agronomic traits.

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Production of recombinant proteins is primarily established in cultures of mammalian, insect and bacterial cells. Concurrently, concept of using plants to produce high-value pharmaceuticals such as vaccines, antibodies, and dietary proteins have received worldwide attention. Newer technologies for plant transformation such as plastid engineering, agroinfiltration, magnifection, and deconstructed viral vectors have been used to enhance the protein production in plants along with the inherent advantage of speed, scale, and cost of production in plant systems. Production of therapeutic proteins in plants has now a more pragmatic approach when several plant-produced vaccines and antibodies successfully completed Phase I clinical trials in humans and were further scheduled for regulatory approvals to manufacture clinical grade products on a large scale which are safe, efficacious, and meet the quality standards. The main thrust of this review is to summarize the data accumulated over the last two decades and recent development and achievements of the plant derived therapeutics. It also attempts to discuss different strategies employed to increase the production so as to make plants more competitive with the established production systems in this industry.

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BACKGROUND: Fusarium wilt disease of banana is one of the most devastating diseases and was responsible for destroying banana plantations in the late nineteenth century. Fusarium oxysporum f. sp. cubense is the causative agent. Presently, both race 1 and 4 strains of Foc are creating havoc in the major banana-growing regions of the world. There is an urgent need to devise strategies to control this disease; that is possible only after a thorough understanding of the molecular basis of this disease. RESULTS: There are a few regulators of Foc pathogenicity which are triggered during this infection, among which Sge1 (Six Gene Expression 1) regulates the expression of effector genes. The protein sequence is conserved in both race 1 and 4 strains of Foc indicating that this gene is vital for pathogenesis. The deletion mutant, FocSge1 displayed poor conidial count, loss of hydrophobicity, reduced pigmentation, decrease in fusaric acid production and pathogenicity as compared to the wild-type and genetically complemented strain. Furthermore, the C-terminal domain of FocSge1 protein is crucial for its activity as deletion of this region results in a knockout-like phenotype. CONCLUSION: These results indicated that FocSge1 plays a critical role in normal growth and pathogenicity with the C-terminal domain being crucial for its activity.

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Bidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5' ends in close proximity. These genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. This intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely ß-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. Characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.

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Pests and pathogens restrict the production potential of many crop plants. The losses incurred due to pests and diseases are huge threatening food security. Management strategies include use of chemical pesticides which can be detrimental to human health and environment and other physical and biological methods which have serious limitations. An alternative would be to utilize the advanced technology such as RNA interference (RNAi) to engineer disease resistance in crop plants. The phenomenon of RNAi is very well studied in organisms across genera and found to be conserved. Taking advantage of this, dsRNAs have been delivered into pests and pathogens and showed significant growth inhibition. Banana is susceptible to various groups of pathogens which results in poor yield. The proof-of-principle studies using RNAi technology have already been demonstrated in banana to develop resistance to two important groups of pathogens. Transgenic banana plants expressing small interfering RNA targeting BBTV and Fusarium pathogen have shown high level of resistance. In this review, we summarize and discuss the studies utilizing RNAi as a strategy to develop resistance to major banana diseases and encourage further research in exploiting RNAi-based resistance in other crop plants.

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Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular.

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Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is counted among the most destructive diseases of crop plants in India. In the absence of any credible control measure to manage this disease, development of resistant cultivars is the best option. Somaclonal variations arising out of long term in vitro culture of plant tissues is an important source of genetic variability and the selection of somaclones having desired characteristics is a promising strategy to develop plants with improved characters. In the present study, we isolated a group of somaclonal variants of banana cv. Rasthali which showed efficient resistance towards Foc race 1 infection in repeated bioassays. cDNA-RAPD methodology using 96 decamer primers was used to characterize these somaclonal variants. Among the four differentially amplified bands obtained, one mapping to the coding region of a lipoxygenase gene was confirmed to be down regulated in the somaclones as compared to controls by real-time quantitative RT-PCR. Our results correlated well with earlier studies with lipoxygenase mutants in maize wherein reduced expression of lipoxygenase led to enhanced resistance towards Fusarium infection.

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In order to feed an ever-increasing world population, there is an urgent need to improve the production of staple food and fruit crops. The productivity of important food and fruit crops is constrained by numerous biotic and abiotic factors. The cultivation of banana, which is an important fruit crop, is severely threatened by Fusarium wilt disease caused by infestation by an ascomycetes fungus Fusarium oxysporum f. sp. cubense (Foc). Since there are no established edible cultivars of banana resistant to all the pathogenic races of Foc, genetic engineering is the only option for the generation of resistant cultivars. Since Foc is a hemibiotrophic fungus, investigations into the roles played by different cell-death-related genes in the progression of Foc infection on host banana plants are important. Towards this goal, three such genes namely MusaDAD1, MusaBAG1 and MusaBI1 were identified in banana. The study of their expression pattern in banana cells in response to Foc inoculation (using Foc cultures or fungal toxins like fusaric acid and beauvericin) indicated that they were indeed differentially regulated by fungal inoculation. Among the three genes studied, MusaBAG1 showed the highest up-regulation upon Foc inoculation. Further, in order to characterize these genes in the context of Foc infection in banana, we generated transgenic banana plants constitutively overexpressing the three genes that were later subjected to Foc bioassays in a contained greenhouse. Among the three groups of transgenics tested, transformed banana plants overexpressing MusaBAG1 demonstrated the best resistance towards Foc infection. Further, these plants also showed the highest relative overexpression of the transgene (MusaBAG1) among the three groups of transformed plants generated. Our study showed for the first time that native genes like MusaBAG1 can be used to develop transgenic banana plants with efficient resistance towards pathogens like Foc.

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Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

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Antimicrobial peptides are a potent group of defense active molecules that have been utilized in developing resistance against a multitude of plant pathogens. Floral defensins constitute a group of cysteine-rich peptides showing potent growth inhibition of pathogenic filamentous fungi especially Fusarium oxysporum in vitro. Full length genes coding for two Petunia floral defensins, PhDef1 and PhDef2 having unique C-terminal 31 and 27 amino acid long predicted prodomains, were overexpressed in transgenic banana plants using embryogenic cells as explants for Agrobacterium-mediated genetic transformation. High level constitutive expression of these defensins in elite banana cv. Rasthali led to significant resistance against infection of Fusarium oxysporum f. sp. cubense as shown by in vitro and ex vivo bioassay studies. Transgenic banana lines expressing either of the two defensins were clearly less chlorotic and had significantly less infestation and discoloration in the vital corm region of the plant as compared to untransformed controls. Transgenic banana plants expressing high level of full-length PhDef1 and PhDef2 were phenotypically normal and no stunting was observed. In conclusion, our results suggest that high-level constitutive expression of floral defensins having distinctive prodomains is an efficient strategy for development of fungal resistance in economically important fruit crops like banana.

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