Ganjun Yi

Transcriptome profiling of resistant and susceptible Cavendish banana roots following inoculation with Fusarium oxysporum f. sp. cubense tropical race 4

BackgroundFusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is considered the most lethal disease of Cavendish bananas in the world. The disease can be managed in the field by planting resistant Cavendish plants generated by somaclonal variation. However, little information is available on the genetic basis of plant resistance to Foc TR4. To a better understand the defense response of resistant banana plants to the Fusarium wilt pathogen, the transcriptome profiles in roots of resistant and susceptible Cavendish banana challenged with Foc TR4 were compared.ResultsRNA-seq analysis generated more than 103 million 90-bp clean pair end (PE) reads, which were assembled into 88,161 unigenes (mean size = 554 bp). Based on sequence similarity searches, 61,706 (69.99%) genes were identified, among which 21,273 and 50,410 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 33,243 (37.71%) unigenes to 119 KEGG pathways. A total of 5,008 genes were assigned to plant-pathogen interactions, including disease defense and signal transduction. Digital gene expression (DGE) analysis revealed large differences in the transcriptome profiles of the Foc TR4-resistant somaclonal variant and its susceptible wild-type. Expression patterns of genes involved in pathogen-associated molecular pattern (PAMP) recognition, activation of effector-triggered immunity (ETI), ion influx, and biosynthesis of hormones as well as pathogenesis-related (PR) genes, transcription factors, signaling/regulatory genes, cell wall modification genes and genes with other functions were analyzed and compared. The results indicated that basal defense mechanisms are involved in the recognition of PAMPs, and that high levels of defense-related transcripts may contribute to Foc TR4 resistance in banana.ConclusionsThis study generated a substantial amount of banana transcript sequences and compared the defense responses against Foc TR4 between resistant and susceptible Cavendish bananas. The results contribute to the identification of candidate genes related to plant resistance in a non-model organism, banana, and help to improve the current understanding of host-pathogen interactions.

Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) Seedlings

Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by proteomic analysis.

The use of GFP-transformed isolates to study infection of banana with Fusarium oxysporum f. sp. cubense race 4

Fusarium oxysporum f. sp. cubense (Foc) is the causal pathogen of Fusarium wilt of banana. To understand infection of banana roots by Foc race 4, we developed a green fluorescent protein (GFP)-tagged transformant and studied pathogenesis using fluorescence microscopy and confocal laser scanning microscopy. The transformation was efficient, and GFP expression was stable for at least six subcultures with fluorescence clearly visible in both hyphae and spores. The transformed Foc isolate also retained its pathogenicity and growth pattern, which was similar to that of the wild type. The study showed that: (i) Foc race 4 was capable of invading the epidermal cells of banana roots directly; (ii) potential invasion sites include epidermal cells of root caps and elongation zone, and natural wounds in the lateral root base; (iii) in banana roots, fungal hyphae were able to penetrate cell walls directly to grow inside and outside cells; and (iv) fungal spores were produced in the root system and rhizome. To better understand the interaction between Foc race 4 and bananas, nine banana cultivars were inoculated with the GFP-transformed pathogen. Root exudates from these cultivars were collected and their effect on conidia of the GFP-tagged Foc race 4 was determined. Our results showed that roots of the Foc race 4-susceptible banana plants were well colonized with the pathogen, but not those of the Foc race 4-resistant cultivars. Root exudates from highly resistant cultivars inhibited the germination and growth of the Fusarium wilt pathogen; those of moderately resistant cultivars reduced spore germination and hyphal growth, whereas the susceptible cultivars did not affect fungal germination and growth. The results of this work demonstrated that GFP-tagged Foc race 4 isolates are an effective tool to study plant–fungus interactions that could potentially be used for evaluating resistance in banana to Foc race 4 by means of root colonization studies. Banana root exudates could potentially also be used to identify cultivars in the Chinese Banana Germplasm Collection with resistance to the Fusarium wilt pathogen.

Contamination of Bananas with Beauvericin and Fusaric Acid Produced by Fusarium oxysporum f. sp. cubense

Background Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis. Methodology/Principal Findings Twenty isolates of Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak ‘Guangfen #1’ and 10 Cavendish ‘Brazilian’ plants. Fusaric acid and BEA were detected in all the tissues, including the fruits. Conclusions/Signficance The current study provides the first investigation of toxins produced by Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.

Rapid screening of Musa species for resistance to Fusarium wilt in an in vitro bioassay

In order to accelerate breeding and selection for disease resistance to Fusarium wilt, it is important to develop bioassays which can differentiate between resistant and susceptible cultivars efficiently. Currently, the most commonly used early bioassay for screening Musa genotypes against Fusarium oxysporum f. sp. cubense (Foc) is a pot system, followed by a hydroponic system. This paper investigated the utility of in vitro inoculation of rooted banana plantlets grown on modified medium as a reliable and rapid bioassay for resistance to Foc. Using a scale of 0 to 6 for disease severity measurement, the mean final disease severities of cultivars expressing different levels of disease reaction were significantly different (P ≤ 0.05). Twenty-four days after inoculation with Foc tropical race 4 at 106 conidia ml−1, the plantlets of two susceptible cultivars had higher final disease severities than that of four resistant cultivars. Compared with ‘Guangfen No.1’, ‘Brazil Xiangjiao’ is highly susceptible to tropical race 4 and its mean final disease severity was the highest (5.27). The plantlets of moderately resistant cultivar ‘Formosana’ had a mean final disease severity (3.53) lower than that of ‘Guangfen No.1’ (4.33) but higher than that of resistant cultivars: ‘Nongke No.1’, GCTCV-119, and ‘Dongguan Dajiao’ (1.87, 1.73, and1.53, respectively). Promising resistant clones acquired through non-conventional breeding techniques such as in vitro selection, genetic transformation, and protoplast fusion could be screened by the in vitro bioassay directly. Since there is no acclimatization stage for plantlets used in the bioassay, it helps to improve banana breeding efficiency.

An efficient protocol for the production of chit42 transgenic Furenzhi banana (Musa spp. AA group) resistant to Fusarium oxysporum

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a destructive fungal disease of banana. Transferring antifungal genes into banana provides a feasible way to control fungal disease, but transformation frequencies for banana are often cultivar and cell line dependent. We investigated an efficient liquid medium selection protocol for an Agrobacterium-mediated transformation system for Furenzhi (Musa spp. AA group). Embryogenic cell suspensions (ECS) of Furenzhi were co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring a plasmid containing the endochitinase gene chit42 from Trichoderma harzianum. After co-cultivation, GUS-positive ECS were selected in liquid medium with antibiotics, and compared to semi-solid medium selection. In total, 186 transgenic plantlets were obtained using M2S liquid medium (based on Murashige and Skoog salts) with hygromycin, whereas no transgenic lines were obtained in parallel experiments with semi-solid selection medium. Integration of the transgene was confirmed by PCR, and Southern blots which showed a single copy of the transgene had integrated into the banana genome in three plant lines. Expression of the transgene in regenerated plants was confirmed by ß-glucuronidase histochemical assays and real-time PCR. Both in vitro and ex vivo disease assays showed that the majority of transgenic lines (three of seven) expressing chit42 showed a higher level of resistance to Fusarium wilt (Foc race 4), whereas non-transgenic control plants were susceptible. These results imply a relationship between Foc4 disease resistance and the transcription levels of the transgene in the transgenic clones. This study may offer a promising approach to breed bananas resistant to the fungal disease Fusarium wilt.

The toxic mechanism and bioactive components of Chinese leek root exudates acting against Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt is one of the most serious threats to banana production worldwide. The disease is caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), especially tropical race 4 (Foc TR4). It has been reported that crop rotation and intercropping of Chinese leek (Allium tuberosum) with banana can effectively reduce incidence of the disease in the field. In this study, we investigated the toxic mechanism and bioactive compounds of Chinese leek root exudates (CLREs) acting against Foc TR4. In vitro experiments showed that CLREs inhibit the germination and growth of Foc TR4, initiate the accumulation of reactive oxygen species (ROS), and trigger a decrease in the mitochondrial transmembrane potential (ΔΨm). In addition, it induced decreases in the expression of ergosterol biosynthesis genes, and up-regulated the expression of genes associated with autophagy. Dimethyl trisulfide, dimethyl disulfide, 2-propenyl methyl disulfide, 2-propenyl methyl trisulfide, and 2-methoxy-4-vinylphenol are major components of CLREs volatiles, and strongly inhibit Foc TR4 development. These results suggest that CLREs induce cell death in Foc TR4 by inducing oxidative bursts, mitochondrial impairment, and plasma membrane depolarization. In addition, sulfur and phenolic compounds contribute to its antifungal activity.

MpMYBS3 as a crucial transcription factor of cold signaling confers the cold tolerance of banana

Banana (Musa spp.) is an important tropical crops. Low temperature is one of the key environmental stresses, which greatly affects the global banana production. Different varieties of banana exhibit a high degree of genetic variability for cold tolerance. Compared with Cavendish banana, Dajiao has superior cold tolerance. Cloning of Dajiao cold-tolerant genes and characterization of their functions could reveal the molecular mechanism of cold tolerance in Dajiao. Our previous comparative transcriptome analysis of cold-sensitive Cavendish banana and cold-tolerant Dajiao identified several cold-tolerance candidate genes in Dajiao. In this study, a Dajiao candidate gene, MpMYBS3 (homolog of MYBS3 in rice), encoding a transcription factor, was cloned and characterized. Amino acid sequence alignments showed that MpMYBS3 belongs to the R1-type MYB transcription factors. Subcellular localization analysis indicated that MpMYBS3 is located in the nucleus. Heterologous overexpression of MpMYBS3 in banana showed that the transgenic lines had significantly higher cold tolerance than the wild-type, which might be associated with the increased accumulation of proline, and a reduction in malondialdehyde content and electrolyte leakage. Surprisingly, MYBS3 repressed the well-known ICE1–CBF-dependent cold signaling pathway in banana: the MaCBF1 and MaCBF2 genes were repressed at the transcriptional level after cold treatment. However, MaMKRY46 was significantly induced in transgenic bananas overexpressing MpMYBS3 under cold stress. These findings suggest that MYBS3-mediated cold signaling as a key player in cold adaptation of banana.

Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana ( Musa spp .) Cold Signal Network

Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism.

Genome-Wide Computational Analysis of Musa Microsatellites: Classification, Cross-Taxon Transferability, Functional Annotation, Association with Transposons & miRNAs, and Genetic Marker Potential

The development of organized, informative, robust, user-friendly, and freely accessible molecular markers is imperative to the Musa marker assisted breeding program. Although several hundred SSR markers have already been developed, the number of informative, robust, and freely accessible Musa markers remains inadequate for some breeding applications. In view of this issue, we surveyed SSRs in four different data sets, developed large-scale non-redundant highly informative therapeutic SSR markers, and classified them according to their attributes, as well as analyzed their cross-taxon transferability and utility for the genetic study of Musa and its relatives. A high SSR frequency (177 per Mbp) was found in the Musa genome. AT-rich dinucleotide repeats are predominant, and trinucleotide repeats are the most abundant in transcribed regions. A significant number of Musa SSRs are associated with pre-miRNAs, and 83% of these SSRs are promising candidates for the development of therapeutic SSR markers. Overall, 74% of the SSR markers were polymorphic, and 94% were transferable to at least one Musa spp. Two hundred forty-three markers generated a total of 1047 alleles, with 2-8 alleles each and an average of 4.38 alleles per locus. The PIC values ranged from 0.31 to 0.89 and averaged 0.71. We report the largest set of non-redundant, polymorphic, new SSR markers to be developed in Musa. These additional markers could be a valuable resource for marker-assisted breeding, genetic diversity and genomic studies of Musa and related species.