Differential expressed genes of banana leaves induced by Fusarium wilt disease  

Peng T.C. , Chen Y.Y. , Luo J.Y. , Zhao H. , Mu L. , Xie J. , Wei S.S. , Xia Y.Q. , Tang H.
National Key Laboratory Base of Tropic Biological Resources Sustainable Utilization, College of Agriculture, Hainan University, Haikou 570228, P.R. China
Author    Correspondence author
Molecular Plant Breeding, 2015, Vol. 6, No. 20   doi: 10.5376/mpb.2015.06.0020
Received: 25 Aug., 2015    Accepted: 26 Sep., 2015    Published: 08 Nov., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Peng T.C., Chen Y.Y., Luo J.Y., Zhao H., Mu L., Xie J., Wei S.S., Xia Y.Q. and Tang H., 2015, Differential expressed genes of banana leaves induced by Fusarium wilt disease, Interaction, Molecular Plant Breeding, 6(20): 1-10 (doi: 10.5376/mpb.2015.06.0020)

Abstract

Fusarium wilt of banana is a destructive fungus disease caused by Fusarium oxysporum f. sp. Cubense. This disease had harmful effects on banana planting and its industrial production. In order to study the pathogenic mechanism of banana Fusarium wilt disease, we used cDNA-AFLP technique to identify differential expressed transcripts that were induced or inhibited by Fusarium oxysporum f. sp. Cubense tropical race 4 infection. Among 223 isolated cDNA fragments, we sequenced and get 137 unique banana cDNAs that involved in different functions, including disease and defense response, transcription regulation, signal transduction, primary metabolism, energy metabolism, cell growth and division, protein destination and storage, and so on. Among these genes, 8 ESTs were related to disease and defense response including NBS-LRR type resistance gene, hot shock protein, and alcohol dehydrogenase; 19 ESTs were related to transcriptional regulation including transcription factor MYB and MYC, zinc finger protein, and Glycine-rich RNA binding protein; 12 ESTs were related to signal transduction including the Ras-related nuclear proteins. We choose out 6 important genes to analyze the expression patterns by sqRT-PCR, and confirm their relationship with pathogenic mechanism of banana Fusarium wilt.

Keywords
Banana; Fusarium oxysporum f. sp. Cubense; Differential expressed genes; Fusarium wilt disease; Pathogenic response

Introduction
Banana is an important tropical monocot crop species as food or fruit. Fusarium wilt of banana, known as Panama disease, is a devastating vascular systemic disease. In recent years, this disease has burst out in banana production regions around the world, has caused great economical loss to farmer, and it is really a great threat to banana production (De Ascensao et al., 2000). Fusarium wilt disease is widely distributed in South Pacific, Asia, Australia, Africa and tropical America; in China, it mainly exists in Guangdong, Guangxi, Yunnan, and Hainan provinces (Wei et al., 2005).
 
The Fusarium wilt of banana is caused by Fusarium oxysporum f. sp. cubense (Foc), a pathogenic soil borne fungus. This fungus infects banana plants through the wound of roots and leaves, and it can be transferred and spread by soil, water and infected suckers (Van Den Berg et al., 2007). Fusarium oxysporum f. sp. cubense has 4 races, the race 4 has the strongest damage on banana production (Thangavelu et al., 2012), and the tropical race 4 (TR4) is the most dangerous fungus in China.

In order to prevent banana Fusarium wilt, the most economical and fundamental pathway is to breed disease-resistant varieties, because chemical control can not reach ideal effects. The discovery of banana defense mechanism is a prerequisite for disease control through molecular breeding. In molecular or transcriptome level, to understand the pathogenic mechanism of banana to Foc will provide a scientific basis to develop effective measures to control disease (Groenewald et al., 2006). Through transcriptomic profile analysis (Li et al., 2012) found that the recognition of PAMPs and defense-related genes may contribute to Foc4 resistance in banana. The sequencing of banana (AA) genome provides great promotion on banana research (D’ Hont et al., 2012). The reason for banana susceptibility to Foc TR4 is that SA biosynthesis-related genes are suppressed and the induced resistance of banana against Foc TR4 might be a case of salicylic acid-dependent systemic acquired resistance(Wang et al., 2015).

However, the molecular pathological mechanism of banana to Foc TR4 is not clear. The objective of this study was to identify some key genes differentially expressed in banana leaves at different time points after Foc-TR4 inoculation by cDNA-AFLP, verify their expression patterns through semi-quantitative RT-PCR, and provide new genetic information for genetic improvement on banana disease-resistance.

Materials and Methods
Biological Materials
Micropropagated Cavendish banana plantlets of the variety ‘Brazilian’ for our experiments was get from banana seedling cultivation factory in Danzhou City, Hainan Province. Fusarium oxysporum f. sp. Cubense tropical race 4 (Foc-TR4) was separated and preserved by our research group.

Pathogen inoculation treatment
Foc-TR4 strains were inoculated in PDA medium (200 g Potato, 20 g Glucose and 18g Agar per liter medium) plate, and cultured in the 25℃ and dark conditions for 4 days. The hyphae of Foc-TR4 picked up by inoculating loop, was inoculated in the CMC liquid medium (7.5 g C8H11O5Na, 0.5 g Yeast extract, 0.5 g NH4NO3, 0.5 g KH2PO4 and 0.25 g MgSO4 per liter medium), and cultured 5 days in 28℃ shaking condition (150 r/min) for conidia growth. The hyphae were filtered by double layer of sterile gauze, the cultured medium suspension was centrifuged for 5min at 12000rpm, discarded the supernatant and the precipitate was conidia. The conidia were suspended in sterilized H2O, detected and counted under 400× ordinary light microscope, and diluted to 1.0×105 spores/mL concentration (early study data) in sterilized H2O.

When the banana plantlets grew to six leaves stage, we cut the health and clean young banana leaves for inoculation experiments. Banana leaves were placed in the 20 cm×35 cm×50 cm plastic box, the petioles of leaves were covered with absorbent paper sprayed enough sterile water. Pinprick six wounded spots evenly on each leaf and inoculate 20 µL Foc-TR4 conidia solution on wounded spots for disease inducing. The experiment set up control and treatment group for comparison, the control group was inoculated with 20 µL sterile water, and the treatment group with 20 µL Foc-TR4 conidia solution. We took samples at 4 different time points after inoculation: 4h, 24h, 3d and 6d, to study the gene differential expression of banana induced by Foc-TR4 infection.
 
RNA preparation and cDNA synthesis
Total RNA was isolated from 200 mg banana leaf tissues with the CTAB extraction method. The CTAB extraction solution contains 2.5% CTAB, 100mM Tris-HCl pH8.0, 1.4M NaCl, 20mM EDTA, pH8.0 and 4% PVP. The steps were as follows. First, weight 200 mg banana leaf tissues and grind fully in liquid nitrogen, add 1mL of 65℃ preheated CTAB extraction and 20 µL β-mercaptoethanol, transfer the homogenate to 2.0 mL eppendorf tubes in 65℃ water bath for 30min, mixing them every 5min. Extract them twice with 0.6mL chloroform, then add 5/8 volume of 8M LiCl to the supernatant, keep in -80℃ refrigerator for 1h, and centrifuge with 12000 rpm for 20min at 4℃. Wash the precipitate with 75% ethanol, dissolve them in 400 μL DEPC-treated water, add 250 μL 8 M LiCl, place them at -20℃ for 4 h, centrifuge with 12000rpm for 20 min at 4℃. Finally, wash RNA with 75% ethanol and dissolve RNA in 50 μL DEPC- treated water. Take 5 μL RNA samples for electrophoresis analysis and concentration determination, adjust the RNA concentration to the same level, and store the RNA in -80℃ refrigerator for following experiments. 2 μg total RNA was used initially for first strand cDNA synthesis for each sample, followed by second strand cDNA synthesis, using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Beijing, China).

cDNA-AFLP experiment
About 200ng double-stranded cDNA was subjected to standard AFLP template production. The restriction enzymes used for cDNA digestion were EcoR I and Mse I (Fermentas, Beijing, China). The digested products were ligated to adapters which sequences were as follows: EcoR I adapter, 5’-CTCGTAGACT GCGTACC-3’ and 5’-AATTGGTACGCAGTCTAC-3’; Mse I adapter, 5’- GACGATGAGTCCTGA G-3’ and 5’-TACTCAGGACTCAT-3’.
The ligated products were pre-amplified with the pre-amplification primers which sequences were as follows: 5’-GTAGACTGCGTACCAATTC-3’, and 5’-GACGATG AGTCCTGAGTAA-3’. Equal amounts of pre-amplified products were selective amplified with the selective amplification primers. The sequences of selective primers were listed in Table 1.
 


Table 1 Sequence of selective amplification primers 


Totally 160 different primer pair combinations were selectively amplified. 6 μL selectively amplified products were mixed with 4 μL formamide dye solution (98% formamide, 10 mM EDTA, 0.05% each of xylene cyanol and bromophenol blue), heat-denatured for 5min at 100℃, and then placed on ice. The denatured mix were loaded onto 6% denaturing polyacrylamide gel and electrophoresed in 1×TBE electrohphoresis buffer at 100 W for 2.5 h. The gels were silver-stained following the procedures as described (Maheswaran et al., 1997). All the reactions for digestion, ligation, pre-amplification, and selective amplification were performed following the procedures as described (Subudhi et al., 1998).

Transcript-derived fragment isolation and re-am- plification
The polymorphic transcript-derived fragments (ESTs) gel bands, selected by presence, absence or differential intensity, were cut from the gel with a sharp blade carefully to avoid any contaminating fragments, and eluted in 100μL sterile double distilled water, then kept at 95℃ for 20min and hydrated overnight at 4℃. 10μL aliquot was used as template for re-amplification in 25μL PCR reaction system, using the same set of corresponding selective primers and the same PCR conditions as selective amplification (Jayaraman et al., 2008). The PCR products were electrophoresed in 1.5% 1×TAE agarose gel, and each single clear band was isolated and eluted using the Bioteke gel extraction kit (DP1721, BioTeke Corporation, Beijing, China).
 
Cloning and sequencing of ESTs
The eluted ESTs were cloned into the plasmid pUCm-T vector (Sangon Biotech, Shanghai, China) following the manufacturer’s protocol; the recombinant plasmid was transformed into DH5α E. coli competent cell. The positive clone was selected out for sequencing in Sangon Biotech Co., Ltd (Shanghai, China).
 
Bioinformatic analysis of sequences
The sequences of the ESTs were analyzed for their homology with the publicly available database, including the Genbank (http://www.ncbi.nlm.nih.gov/blast) and The Banana Genome Hub (http://banana- genome.cirad.fr/blast) using BLASTn and BLASTx algorithms, to get the useful bioinformation about the ESTs.

Semi-quantitative RT-PCR
Reverse transcription-PCR was performed from banana total RNA using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Beijing, China) according to the manufacture’s manual. Primers were designed from the sequences of the ESTs using Primer5.0 software. The house-keeping gene 18S rDNA was used as the internal standard for quantity and quality checking of RNA template in RT-PCR. The PCR condition was essentially same as described earlier for re-amplification of ESTs, with corresponding anneal temperature for different primer pairs as listed in Table 2.
 


Table 2 Primers for semi-quantitative RT-PCR of ESTs 


Results
Disease infection to banana leaves
Banana leaves from uniform seedlings were cut for in vitro pathogen inoculation experiment. Foc-TR4 conidia was inoculated on leaves as infection treatment, the sterile H2O was inoculated as control treatment. The leaves of infection treatment showed typical disease symptoms of Fusarium wilt disease at 3 dpi (days post inoculation); the lesion expanded on leaves with yellow color changes. At the same time, the control treatment showed no disease symptom (Figure 1).
 


Figure 1 The pathogenic symptom of banana leaves at 3 dpi. The left leaf was inoculated with Foc-TR4 as infection treatment, the right leaf inoculated with sterile H2O as control. 

 
Screening of differential expressed ESTs by cDNA-AFLP
High quality RNA of 4 different time points (4 h, 24 h, 3d and 6 d after inoculation) were extracted for cDNA-AFLP experiment. A total of 256 primer combinations were used for cDNA-AFLP analysis from both control and infection treatment. On average,60-80 clear and unambiguous bands (ESTs) were generated by each primer combination, which yielded more than 10,000 bands totally. There were 223 differentially expressed ESTs isolated from silver-stained cDNA-AFLP polyacrylamide gels (Figure 2), according to their presence/absence, or quantitative difference between the infection treatment and control at different time points. All isolated EST fragments were eluted from the polyacrylamide gel, and these fragments were reamplified by selective amplification condition.
 


Figure 2 The cDNA-AFLP products of three primer combina- tions on PAGE gel. 


Sequence analysis of ESTs
223 re-amplified ESTs were linked to pUCm-T vector and transformed into E. coli competent cell. The positive clones were sequenced and compared with bioinformatic database including Genbank of NCBI and The Banana Genome Hub (http://banana- genome.

cirad.fr/) by BLAST program, as results, 137 unique ESTs were found. Most of ESTs had homology to genes with known functions (Figure 3), whereas, 18% of them were unclear classification, and 17% were new EST with no similarity to any known function genes. According to the functional catalogues, among the known function ESTs, 7% belonged to disease defense, 10% belonged to signal transduction, 13% belonged to transcription, and so on. So presumably, the molecular regulation mechanism of banana responded to Foc- TR4 disease was very complicated.
 


Figure 3 Functional catalogues distribution of ESTs derived from the cDNA-AFLP 


The special genes related to disease and defense response
When plant suffered from pathogenic stress or infection, its first response is to stimulate the cell defense response system to react to pathogen. The receptor on cell membrane will receive and transfer infection signal into cell, it will activate some genes or suppress other genes. How banana is responded to Fusarium oxysporum f. sp. Cubense infection? It is really an important and complicated question needed to discover. In this research, 8 ESTs (Table 3) were classified into the functional catalogue of disease and defense responses. These genes included in NBS-LRR type resistance protein, heat shock protein, alcohol dehydrogenase, and so on.
 


Table 3 ESTs related to disease and defense response 


The NBS-LRR type resistance protein has a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. LRR has a close relationship with different proteins interaction and signal processes, and exists in variety proteins whose function and cellular localization are different. The NBS has an N-terminal sub-domain that contains consensus kinase 1a (P-loop), kinase 2 and kinase 3a motifs common to a large variety of nucleotide proteins (Traut et al., 1994). Many plant disease resistance genes have a great many of NBS resistance genes which contain a highly conserved amino acid region, which is closely associated with pathogenic recognition and defense response. Consequently, the NBS-LRR genes provide very effective strategies and methods for exploring, identifying and using the unknown plant disease resistance gene analogues. Up to now, many DNA and cDNA sequences that have homology with the known resistance genes had been isolated from Arabidopsis, soybean, lettuce, tobacco, potato, barley, wheat, rice, tomato, apple and citrus (Yoshimura et al., 1998; Ori et al., 1997; Lee et al., 2003; Deng et al., 2003). In this study, the banana NBS-LRR resistant gene (E113) was isolated and its quantitative expression pattern showed significant up-regulation after Foc-TR4 infection on leaves, which indicated the NBS-LRR type resistance gene may play a certain role in banana Fusarium wilt disease prevention.

Heat shock protein (HSP) is a kind of stress-activated factor. It is induced by various abiotic and biotic stresses, such as water stress, salt stress, hypoxia, ABA, nitrite, cadmium, and virus, and so on. The diversity of induction factors indicated that plants had cross-resistance with different stresses and had some common molecular basis within a variety of adversity stresses. The heat shock protein had been found from barley, wheat, millet, soybean, carrot, tomato, cotton, tobacco, Arabidopsis, and so on. Plants must cope with various acute environmental changes including biotic and abiotic stresses. Proteins can be misfolded and aggregate under stress and lead to many functional problems in cell, and the misfolded protein should be refolded to recover the right conformation. HSPs are molecular chaperones and protein remodeling factors, some of them are upregulated in response to stress (Wang et al., 2004). Hsp70 gene in Trichoderma harzianum conferred tolerance to heat and other abiotic stresses to this fungus. Transgenic Arabidopsis thaliana plants expressing the T. harzianum hsp70 gene exhibited enhanced tolerance to heat stress; hsp70 confers tolerance to heat and other abiotic stresses, and the fungal HSP70 protein acts as a negative regulator to HSF transcriptional activity in Arabidopsis (Montero-Barrientos et al., 2010). In this study, the banana hsp70 gene (E121) was isolated and showed significant down-regulation after Foc-TR4 infection on leaves, hsp70 gene may confer tolerance to banana Fusarium wilt disease.

The alcohol dehydrogenase (ADH) is a metalloenzyme containing zinc, has a wide range of substrate specificity, and widely exists in human, animal liver, plants and microbial cells. ADH plays an important role in the anaerobic respiration process in plant. Some studies showed that ADH belonged to the protein induced by anaerobic, the activity of ADH in root tissue could greatly increase when terrestrial plant was under the condition of waterlogged soil or anaerobic environment. After waterflooding, the short-term increase of its activity was good for energy supply of plant (Ning et al., 2009). After plant is infected by pathogen, the organism will gradually lose vitality, the metabolic activity also will gradually weaken and respiration will decrease. By this time, the activity of ADH will rise, which can supply energy for plant momently.

The special genes relevant to transcriptional regulation
Transcriptional regulation is the most important regulation for plant gene expression. The special expression of genes is largely regulated on transcriptional level. When plant is adapted to pathogen infection, plant obtains a complex defense response mechanism. After plant identifies pathogen, the related gene expression of plant defense response is finally induced through a series of signal transduction (Meng et al., 2013). In this research, 19 ESTs (Table 4) might be related to transcriptional regulation. These genes came down to a variety of transcriptional regulation pathway, consisting of transcription factor of MYB and MYC, zinc finger protein, retrotransposon, Glycine-rich RNA binding protein, and so on.
 


Table 4 ESTs related to transcriptional regulation 


The transcription factors (TFs) play an important role in the regulating gene expression by activating or suppressing the gene expression, such as plant growth and development, morphogenesis, as well as its response to environment. Plant TFs control gene expressions and genes control many physiological processes, which in turn trigger cascades of biochemical reactions in plant cells. Studies had shown that the plant transcription factors that played an important role in plant stress-resistance response could regulate the expression of genes related to pathogen, drought, high salinity, low temperature, hormone, and so on. Now, there are 129, 288 plant transcription factors identified from 83 species and classified them into 58 families (Zhang et al., 2011). According to the difference of the conservative DNA-binding domain, the transcription factors is divided into the following catrgories: MYB、AP2、NAC、MYC、HD、C2H2(Zn)、ARF、WRKY, and so on.

In the family of transcription factor, MYB is the most one and the function of MYB is the most diverse. They play an important role in regulating plant stress responses. When the plant is forced by biological stress including pathogen or insect damage, for example, the overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops (Liu et al., 2013). Hypersensitive response (HR) is a cell programmed death process related to plant resistance to diseases. Salicylic acid (SA) plays an important role in resisting pathogen infection. When plant is induced by pathogens, plant has two main types of systemic resistance, one is system acquired resistance (SAR) dependent on salicylic acid, and for example, AtMyb30 in Arabidopsis is a MYB transcription factor gene of special transient expression in the early HR. Through the transgenic experiment for AtMyb30 of overexpression and suppression in Arabidopsis and tobacco, it turned to be AtMyb30 acts as a positive regulator of the HR (Vailleau et al., 2002). AtMyb30 expression in response to an HR-inducing bacterial pathogen is dependent on SA accumulation, but NPR1-independent, and alterations of AtMyb30 expression modulate SA levels and SA-associated gene expression (Raffaele et al., 2006). The other is induced systematic resistance (ISR) dependent on jasmonic acid (JA) and ethylene (ET). Some MYB transcription factors may be activated in signal molecules JA and defense response, which in turn regulates later resistance-associated gene overexpression, and reduces the damage of pathogens.

The MYC transcription factor also is an important type in transcription factor family. The study showed that AtMYC2 could be induced rapidly by exogenous ABA or drought treatments, and AtMYC2 could interact with AtMYB2 (Abe et al., 2003). AtMYC2 differentially regulates the expression of two groups of JA-induced genes. The first group includes genes involved in defense responses against pathogens and is repressed by AtMYC2. The second group, integrated by genes involved in JA-mediated systemic responses to wounding, is activated by AtMYC2 (Lorenzo et al., 2004).

The zinc finger proteins are a super family of proteins involved in numerous activities of plant growth and development and are also known to regulate resistance mechanism for various biotic and abiotic stresses (Feurtado et al., 2011). Because of its special finger-like structure, the zinc finger protein plays an important role in the identification of DNA, protein and RNA, in addition to transcription factors and protein adapter, some zinc finger proteins also have RNA binding properties and regulate the transcription of target genes. There were thirty four zinc finger domains identified in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids (Gupta et al., 2012). In our study, the transcription factor of MYB and MYC, zinc finger protein and other genes-associated were found. This suggests that MYB, MYC and zinc finger protein are involved in the response and regulation of the interaction between banana and Foc-TR4.

The special genes relevant to signal transduction
When banana is infected by Foc-TR4, how the pathogenic infection signal is transferred into cell, which is one of the great concerns of this study. The results showed that 12 ESTs might be related to signal transduction (Table 5).

Ubiquitin-conjugated E2 enzyme (UBE2) is one of the main components of the proteasome degradation cascade. Previous studies have shown an increase of expression levels in individuals challenged to some pathogen organism such as virus and bacteria. In the process of the ubiquitination, the main enzymes contain ubiquitin-activating enzyme E1, ubiquitin- conjugating enzyme E2 and ubiquitin-protein ligase E3. These enzymes play an important role in maintaining the balance of generation and degradation of intracellular protein, the cell homeostasis and normal function, and so on (Markson et al., 2009). UBE2 protein has a very important relationship with stress resistance. Drought, High temperature, heavy metals and other abiotic stress can induce the expression of ubiquitin-conjugating enzyme E2 in plant, which in turn enhances the adaptive ability to adverse environment.

There were about ten ESTs that may relate to signal trusduction showed in table 5. We compared and analyzed these genes with Banana Genome Hub, Genbank, and KEGG Pathway, but we can’t get clear conclusion about how the banana carried out the signal transduction responded to Fusarium wilt disease infection.
 


Table 5 ESTs related to signal transduction 

 
Gene expression verification by sqRT-PCR
We choose six ESTs belonged to different functional catalogues to analyze the expression patterns (Table 6) by sqRT-PCR to validate the reliability of cDNA- AFLP experiment, and quantitatively assessed the relative abundance of ESTs in banana leaf between the control and infection treatments at four different time points (Figure 4). As results, the quantitative expression patterns (increase or decrease in RNA level) of the 6 ESTs were similar in both cDNA-AFLP and sqRT- PCR experiment, it indicated that cDNA-AFLP was an effective method to identify differential expressed genes.
 


Table 6 The ESTs verified by sqRT-PCR 

 


Figure 4 Semi-quantitative RT-PCR showed differential gene expression of banana leaf between the control and infection treatments at different stages. Lane 1, 2, 3 and 4 were 4 h, 24 h, 3d, 6d time points in control treatment; lane 5, 6, 7 and 8 were 4 h, 24 h, 3 d, 6 d time points in infection treatment. 

 
Discussion
In this study, the interaction mechanism was done by cDNA-AFLP. After analysis of cDNA-AFLP, 223 differentially expressed fragments were obtained. Sequencing and analyzing these fragments, 137 kinds
of ESTs were got totally, among which 64.96% had significant homology with known genes, including 12.41% related to primary metabolism, 5.84% related to energy metabolism, 2.92% related to protein synthesis, 5.11% related to protein destination and storage, 13.87% related to transcription, 4.38% related to transporters, 2.19% related to cell growth and division, 2.19% related to cell structure, 9.48% related to signal transduction, 6.57% related to disease and defense response; the remaining 35.04% ESTs were unknown about its functions, including 24 new-found ESTs.

In this research, 8 ESTs related to disease and defense response were obtained, including NBS-LRR type resistance gene, hot shock protein, alcohol dehydrogenase, and so on; 19 ESTs related to transcriptional regulation, including the transcription factor MYB and MYC, zinc finger protein, Glycine-rich RNA binding protein, and so on; 12 ESTs related to signal transduction, including the ubiquitin-conjugating enzyme E2 and so on; the remains were related to primary metabolism, energy metabolism, and so on.

All in all, the pathological response of banana to Fusarium wilt disease was a complex mechanism, although the genome of doubled-haploid (2n=22, 1C=523 Mb) from the species Musa acuminata (A genome) subspecies malaccencis had been sequenced (D'Hont et al., 2012) and provided great promotion for banana research, more researches are needed to do deeply to elucidate lots of confusing questions.  The results of this study will provide some clues to reveal the molecular mechanism of banana infected by Fusarium oxysporum f. sp. Cubense tropical race 4.

Acknowledgements
This research was supported by the following grants: 30860149 (National Natural Science Foundation of China, NSFC), 31360364(National Natural Science Foundation of China, NSFC), 210172 (Key Scientific Research Program from the Ministry of Education of China), lhxm-2012-2 (Joint Support Program from Tropical Crop Breeding Engineering Center of Ministry of Education and National Crop Science Key Discipline of China), and ZDZX2013023 (Key Scientific Research Program from Hainan Province).

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