Qiaosong Yang

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.

Suppression of glycolate oxidase causes glyoxylate accumulation that inhibits photosynthesis through deactivating Rubisco in rice

Glycolate oxidase (GLO) is a key enzyme for photorespiration in plants. Previous studies have demonstrated that suppression of GLO causes photosynthetic inhibition, and the accumulated glycolate with the deactivated Rubisco is likely involved in the regulation. Using isolated Rubisco and chloroplasts, it has been found that only glyoxylate can effectively inactivate Rubisco and meanwhile inhibit photosynthesis, but little in vivo evidence has been acquired and reported. In this study, we have generated the transgenic rice (Oryza sativa) plants with GLO being constitutively silenced, and conducted the physiological and biochemical analyses on these plants to explore the regulatory mechanism. When GLO was downregulated, the net photosynthetic rate (Pn) was reduced and the plant growth was correspondingly stunted. Surprisingly, glyoxylate, as a product of the GLO catalysis, was accumulated in response to the GLO suppression, like its substrate glycolate. Furthermore, the glyoxylate content was found to be inversely proportional to the Pn while the Pn is directly proportional to the Rubisco activation state in the GLO-suppressed plants. A mathematical fitting equation using least square method also demonstrated that the Rubisco activation state was inversely proportional to the glyoxylate content. Despite that the further analyses we have conducted failed to reveal how glyoxylate was accumulated in response to the GLO suppression, the current results do strongly suggest that there may exist an unidentified, alternative pathway to produce glyoxylate, and that the accumulated glyoxylate inhibits photosynthesis by deactivating Rubisco, and causes the photorespiratory phenotype in the GLO-suppressed rice plants.

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.

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.

Early Cold-Induced Peroxidases and Aquaporins Are Associated With High Cold Tolerance in Dajiao (Musa spp. ‘Dajiao’)

Banana is an important tropical fruit with high economic value. One of the main cultivars (‘Cavendish’) is susceptible to low temperatures, while another closely related specie (‘Dajiao’) has considerably higher cold tolerance. We previously reported that some membrane proteins appear to be involved in the cold tolerance of Dajiao bananas via an antioxidation mechanism. To investigate the early cold stress response of Dajiao, here we applied comparative membrane proteomics analysis for both cold-sensitive Cavendish and cold-tolerant Dajiao bananas subjected to cold stress at 10°C for 0, 3, and 6 h. A total of 2,333 and 1,834 proteins were identified in Cavendish and Dajiao, respectively. Subsequent bioinformatics analyses showed that 692 Cavendish proteins and 524 Dajiao proteins were predicted to be membrane proteins, of which 82 and 137 differentially abundant membrane proteins (DAMPs) were found in Cavendish and Dajiao, respectively. Interestingly, the number of DAMPs with increased abundance following 3 h of cold treatment in Dajiao (80) was seven times more than that in Cavendish (11). Gene ontology molecular function analysis of DAMPs for Cavendish and Dajiao indicated that they belong to eight categories including hydrolase activity, binding, transporter activity, antioxidant activity, etc., but the number in Dajiao is twice that in Cavendish. Strikingly, we found peroxidases (PODs) and aquaporins among the protein groups whose abundance was significantly increased after 3 h of cold treatment in Dajiao. Some of the PODs and aquaporins were verified by reverse-transcription PCR, multiple reaction monitoring, and green fluorescent protein-based subcellular localization analysis, demonstrating that the global membrane proteomics data are reliable. By combining the physiological and biochemical data, we found that membrane-bound Peroxidase 52 and Peroxidase P7, and aquaporins (MaPIP1;1, MaPIP1;2, MaPIP2;4, MaPIP2;6, MaTIP1;3) are mainly involved in decreased lipid peroxidation and maintaining leaf cell water potential, which appear to be the key cellular adaptations contributing to the cold tolerance of Dajiao. This membrane proteomics study provides new insights into cold stress tolerance mechanisms of banana, toward potential applications for ultimate genetic improvement of cold tolerance in banana.

Germplasm screening of Musa spp. for resistance to Fusarium oxysporum f. sp. cubense tropical race 4 ( Foc TR4)

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) has seriously threatened global banana production. Recently, identification of Foc TR4 in the Caribbean and Mozambique has aroused widespread concern because it may be directly related to food security for millions of people in Africa and Latin America. To identify a resistant or highly resistant germplasm, 129 accessions were evaluated for Foc TR4 resistance in greenhouses and 100 of them were also assessed in the field. In total, 10 accessions were highly resistant (HR) to Foc TR4; these include four from the AA group, two from the BB group, one of the East African highland bananas (EAHBs), two plantains, and one from wild relatives of banana. Of these, Pahang (AA), Calcutta 4 (AA), and Musa itinerans exhibited the highest degree of resistance with an index of disease (ID2) less than 10, and Foc TR4 could not infect any of them in the field. Furthermore, 31 accessions from AA, AB, AAB, AAAB, and ABB groups and wild relatives were identified as a resistant (R) germplasms. All tested EAHBs and plantains exhibited resistance or high resistance to Foc TR4 infection. Our results provide valuable genetic resources for banana breeding and for studying the mechanisms underlying Fusarium wilt resistance. Furthermore, evaluation of EAHBs and plantains provided a rational for local banana producers and researchers to grow EAHBS and plantains.

Overexpression of Arabidopsis CBF1 Gene in Transgenic Furenzhi banana ( Musa spp. AA group) Improves Resistance to Low Temperature

Cold is an environmental factor that limits the growing season of banana and adversely affects fruit quality and productivity. C-repeat/dehydration responsive element binding factor 1 (CBF1) play important roles in the responses of plants to low-temperature and cold. To test the function and potential use of cold-induced factor CBF1 in improving the chill tolerance of banana, AtCBF1 driven by a cauliflower mosaic virus 35S promoter was introduced into banana plants by Agrobacterium -mediated transformation of embryogenic cell suspensions (ECSs) of banana cultivar Furenzhi (AA). The presence of AtCBF1 transgenes in regenerated plants was confirmed by polymerase chain reaction (PCR) and Southern blots. s-glucoronidase (GUS) histochemical assays reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analysis demonstrated that foreign genes were stably expressed in regenerated plants. Transgenic banana showed growth retardation, thicker leaves and higher chlorophyll content than non-transformed plants. While relative electrolyte leakage (REL) and malondialdehyde (MDA) content were significantly lower in transgenic banana plants than in non-transformed plants under low temperature stress. An obviously higher cold tolerance was observed among the transgenic plants in the cold detection. These results suggest that over-expression of AtCBF 1 in transgenic banana plants plays an important role in improving tolerance to low-temperature.