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In-Silico Identification of Novel Resistant Genes for Fungal Pathogen Fusarium oxysporum f. sp. cubense Race 4: Causative Agent of Banana Vascular Wilt Disease

Shumaila Azam1,2,3*, Anum Munir1,2,3*, Muhammad Saad Khan2, Sahar Fazal2, Azhar Mehmood3, Sartaj Ali3 and Shahid Hussain2

1Bioinformatics International Research Club, Abbottabad, Pakistan

2Departments of Bioinformatics and Biosciences, Capital University of Science and Technology, Islamabad, Pakistan

3Department of Bioinformatics, Government Post Graduate College Mandolin, Abbottabad, Pakistan

*Corresponding Author:
Shumaila Azam
Bioinformatics International Research Club
Abbottabad, Pakistan
Tel: +923348958178
E-mail: [email protected]
 
Anum Munir
Bioinformatics International Research Club
Abbottabad, Pakistan
Tel: +923335852512
E-mail: [email protected]

Received date: December 22, 2016; Accepted date: January 03, 2017; Published date: January 09, 2017

Citation: Azam S, Munir A, Khan MS, Fazal S, Mehmood A, et al. (2017) In-Silico Identification of Novel Resistant Genes for Fungal Pathogen Fusarium oxysporum f. sp. cubense Race 4: Causative Agent of Banana Vascular Wilt Disease. J Plant Biochem Physiol 5: 175. doi: 10.4172/2329-9029.1000175

Copyright: © 2017 Azam S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Cavendish, the most widely grown banana cultivar, is relatively resistant to Race 1 of Fusarium oxysporum f. sp. cubense (Foc1) that caused widespread Panama disease during the first half of the 20th century but is susceptible to Tropical Race 4 of the Foc (Foc TR4), a threat to world banana production. Foc TR4 can spread into the vascular system of banana roots during the early infection process. The genome of the diploid species Musa acuminata; the ancestor of majority of the triploid banana cultivars has recently been sequenced. The identification for the resistant gene in this cultivar is a challenge for the researchers as the resistant gene against Foc4 has not been identified, the major focus of this research work was to identify the pathogenic genes and some resistant genes against those pathogenic genes, to get some better results for the laboratory work. The four resistant genes I, I-1, I-2, and I-3 were identified which provides resistance against Foc4 in banana. About three pathogenic genes SIX1 (SIX1A, SIX1B, SIX1C), SIX2, and SIX8 of Foc4 were identified. Out of these pathogenic genes SIX8 showed the greatest number of interactions with the resistant proteins. So it is proposed that I-1, I-2 and I-3 genes can be used in the further research work for providing better improvements of the resistive cultivars in banana.

Keywords

SIX; Resistance; Pathogenecity; Foc4; Banana; Fol

Introduction

Fusarium wilt of banana (Panama infection), brought about by the soil borne Fusarium oxysporum f. sp. cubense (Foc), is a common disease in the banana (Musa spp.) generation inclusively [1-3]. Foc can affect the several types of Musa and Heliconia, their strains have been arranged into four physiological races. Race1 is pathogenic to ‘Gros Michel’(aaa) and “Silk” (AAB) [4]; Race 2 just influences the crossbreed triploid Bluggoe (ABB) [5], and the race 4 attacks Cavendish cultivars, and all the cultivars susceptible to races 1 and 2, viewed as the most vital on the fact that it influences the cultivars which deliver more than 80% of the world’s bananas [3,6]. The race 4 segregates are subdivided into subtropical race 4 (St4) and tropical race 4 (Tr4). The St4 segregate causes disease in Cavendish bananas in the subtropics [6-8], and Tr4 isolates are pathogenic both under tropical and subtropical conditions [9-12]. In South China, Fusarium wilt of Xiang Jiao (AAA, Cavendish bananas) was initially reported in Guangdong Province in 2001 [13], which brought about by Tr4 [14]. Till date, there are fewer fungicides accessible to control Fusarium wilt of banana. Chemical control is troublesome in light of the fact, that the chlamydospores can make holes in the soil. The best alternative is planting resistant cultivars, for example, Fusarium wilt-resistant bananas chose by means of genetic variations from tissue [15], and transgenic bananas [16,17]. Notwithstanding, Fusarium wilt of banana is still a significant danger to banana production around the world. Quarantine policies and Foc free tissue culture planting materials are the vital methodologies to counteract the spread of infection [18].

The asexual fungus Foc produces three kinds of asexual spores including macroconidia, microconidia and chlamydospore in its life cycle, empowering it to scatter and survive. It imparts a comparable disease cycle to F. oxysporum f. sp. lycopercisi (Fol) thus, brings about tomato wilt disease. Firstly, Foc conidia develop and form fungal hyphae under different supplement conditions and in the host plant environment. Further, fungal hyphae spread around and colonize at the surface of the root. After that, the fungal hyphae would cross the epidermis, attack and colonized the xylem vessels of the root. After effectively contaminating banana roots, the pathogen develops at the rhizome and pseudo stem, causes the demise of the tissue or the whole plant. Lastly, the fungal hyphae and spores on the debris of the banana plant might fall into the soil through rainwater and restart another contamination cycle. Tenuously, disease and colonization of banana plants by the fungal pathogen dependably bring about the wilting and yellowing of the lower parts of the leaves. Internally, discoloration of rhizomes and necrosis of vascular bundles in pseudo stem can be observed in seriously infected banana plants.

As a saprophyte, Foc can persevere in soil for quite a while. When it perceives and sees the stimuli from host plants, it starts polluting host bananas from the area of roots. Few powerful alternatives for dealing with this ineradicable pathogen, as fungicides are generally ineffectual [3]. In this manner, figuring successful control systems for fusarium wilt of bananas is a thing of extraordinary desperation and obliges better understanding of the fungal pathogen, particularly its genome. IN the earlier years, the genomes of the tomato pathogen Fol and the maize pathogen F. verticillioides were sequenced, the Fusarium similar genomics highlights the ancestry genomic locales in Fol that are in charge of the polyphyletic root of host specificity [4].

Materials and Methods

An in vitro investigation of the physiological phenotypes of tomato suggested that recognition of I genes in the tomato are the vital events for vigorous defense, fungal development inhibition, induction of Peroxidase in the in vitro dual culture and ion leakage induced by the cultural filtrates of the Fol pathogen can be markers of resistance [19].

The study of the genomic similarity between the Foc4 and Fol

Foc4 shares a similar infection cycle with F. Oxysporum f. sp. lycopersici (Fol) creating tomato wilt ailment, for the determination of genomic similarity between Foc4 and Fol phylogenetic analysis was done by the help of the phylogeny (FR tool) [20].

Identification of the pathogenic regions in Fusarium oxysporum f. sp. cubense Race 4

The information about the pathogenic SIX1a, SIX1b, SIX1c, SIX2 and SIX8 proteins of the fungus were obtained through literature [21].

Idenification of the resistant genes for the Fusarium oxysporum f. sp. cubense Race 4

The resistant genes of tomato; I, I-1, I-2, I-3 against the pathogenic proteins were identified [22]. These resistant genes can be proposed for the resistant in banana against Foc4 as both Foc4 and Fol pathogens have the same cycle of infection in the hosts.

Sequence retrieval

The amino acid sequences of pathogenic SIX1, SIX2, and SIX8 proteins were retrieved through NCBI database. The protein sequences of resistant I, and I-2 genes were retrieved through UniprotKb and I-1 and I-3 were retrieved through Sol Genomics Network database [23].

3D Models generation

3D models of all the retrieved protein sequences were built by the phyre2 server and validated by Errat server.

Docking of resistant protein structures with pathogenic protein structures

The resistant I, I-1, I-2, I-3 proteins were docked with SIX, SIX2, and SIX8 proteins one by one in Autodock Vina and their amino acid interactions were analyzed in discovery studio software.

Results and Discussion

Through the literature certain characteristics of Foc4 like that of Fol have been identified. These are a) the similarity in the genome, confirmed by phylogenetic analysis shown in Figure 1(b). The similarity in the infection cycle, c) the similar genes that are involved in the pathogenicity.

plant-biochemistry-physiology-Thehylogenetic-genomes-Foc

Figure 1: Thehylogenetic tree that represents similarity in the genomes of Foc race4 (F. oxysporum f. sp. cubense race 4) and Fol (F. oxysporum f. sp. lycopersici).

Here, the focus was to identify the resistant genes against pathogenic proteins and to perform computational analysis of the functionality of the resistant proteins with the assistance of docking procedure, and the interactive analysis of these pathogenic and resistant proteins. The SIX1, SIX2 and SIX8 genes were identified as pathogenic genes and I, I-1, I-2 and I-3 were identified as resistant genes. The 3D structures developed for all the proteins of pathogenic and resistant genes are shown in Figures 2 and 3.

plant-biochemistry-physiology-Pathogenic-proteins-identified

Figure 2: The 3D models of Pathogenic proteins identified for Foc race4 a)SIX1 pathogenic protein b) SIX2 pathogenic protein c) SIX8 pathogenic protein.

plant-biochemistry-physiology-resistant-proteins-Foc

Figure 3: The 3D models of resistant proteins suggested against the Foc race4 a) I resistant protein b) I-1 resistant protein c) I-2 resistant protein d) I-3 resistant protein.

Several in vitro studies have performed by research for the identification of Foc4 as a pathogen of the banana [24,25]. Saraswathi et al. [26] conducted an in vitro experiment for the identification of Fusarium wilt by using fusaric acid and culture filtrate. Late inquires about on Fol, the causal agent of Fusarium wilt of tomato, have elucidated the roles of a few SPs in pathogenicity in the Fol-tomato pathosystem [23]. The SIX proteins SIX1 (Avr3), SIX3 (Avr2) and SIX4 (Avr1) work as either Avr protein (effector) included in the inconsistent cooperation or destructiveness components involved in the perfect associations among tomato and Fol [23]. Scientists are keen on looking Foc4 congregations to distinguish the orthologs of the SIX-coding genes, in particular SIX1-SIX8. The researches have revealed that three orthologs of SIX1 are also found in Foc4 genome, namely SIX1a-SIX1c. Also, Foc4 has one copy of SIX2, SIX6 and SIX8. Furthermore, the Foc4 contains SIX2 and SIX8 genes that are truant in different races of Foc, hence it is further accepted that SIX2 and SIX8 genes may have roles in the infection of Cavendish banana “Brazil”.

Usually, the resistant varieties are generally created by resistant wild types and existing cultivars developed for their properties like great taste, shape and shade. Reproducing of resistant cultivars is an alternative approach to chemical treatment, limiting environmental and consumer risks. Four race-specific R genes for resistance to this pathogen have been genetically mapped in tomato and introgressed into commercial tomato cultivars from wild tomato species [27-34]. The genes I-1 and I-3 are located on chromosome 7, whereas I and I-2 are known to be on the short and long arms of chromosome 11, respectively. The gene I-2 confers resistance to Fol race 2.

The docking studies done to determine the interaction among both resistant and pathogenic proteins further confirmed that, the I, I-1, I-2 and I-3 can be used as resistant genes against Foc race 4. The docking results are shown in Figures 4-6.

plant-biochemistry-physiology-docking-resistant-genes

Figure 4: The docking results of Six1 protein of FOC4 and I resistant genes of FOL, the red ribbons represent the ligand whereas blue ribbons represents the receptors. The binding region between two protein structures is represented by the inter-protein surface. a) Six1-I docked complex b) Six1-I-1 docked complex c) Six1-I-2 docked complex d) Six1-I-3 docked complex.

plant-biochemistry-physiology-docking-resistant-genes

Figure 5: The docking results of Six2 protein of FOC4 and I resistant genes of FOL, the red ribbons represent the ligand whereas blue ribbons represents the receptors. The binding region between two protein structures is represented by the inter-protein surface. a) Six2-I docked complex b) Six2-I-1 docked complex c) Six2-I-2 docked complex d) Six2-I-3 docked complex.

plant-biochemistry-physiology-docking-protein-resistant

Figure 6: The docking results of Six8 protein of FOC4 and I resistant genes of FOL, the red ribbons represent the ligand whereas blue ribbons represents the receptors. The binding region between two protein structures is represented by the inter-protein surface. a) Six8-I docked complex b) Six8-I-1 docked complex c) Six8-I-2 docked complex d) Six8-I-3 docked complex.

Docking strategies authorizes the investigators to monitor a database of compounds and foresees the robust inhibitors in the light of diverse scoring functions [35,36]. Similarly, Morris and Lim stated that Molecular docking is a vigorous tool in the fields of the computeraided drug design and structural molecular biology. The purpose of docking is to anticipate the major binding modes of a ligand with a recognised 3D structure of a protein [34].

The surface is usually designed in the docked complexes to determine the antigenicity of proteins and to analyze the regions of hydrophobicity between a protein molecule. Such a method can appropriately locate the major antigenic sites on the surface proteins of most well characterized infectious organisms [35].

The bonds formed between the pathogenic proteins and resistant protein complexes are shown in Table 1.

SIX1-I, SIX2-I, SIX8-I Hydrogen bonds
Salt bridges
Pi donor bonds
Pi cation bonds
Pi anion bonds
Pi sigma bonds
Pi sulfur bonds
Stacked pi pi bonds
T shaped pi bonds
Amide pi bonds
Pi Alkyl  bonds
Alkyl alkyl bonds
SIX1-I-1, SIX2-I-1, SIX8-I-1 Hydrogen bonds
Salt bridges
Pi donor bonds
Pi cation bonds
Pi anion bonds
Pi sigma bonds
Pi sulfur bonds
Stacked pi pi bonds
T shaped pi bonds
Amide pi bonds
Pi Alkyl  bonds
Alkyl alkyl bonds
Sulfur bonds
SIX1-I-2, SIX2-I-2, SIX8-I-2 Hydrogen bonds
Pi donor bonds
Pi cation bonds
Pi anion bonds
Pi sigma bonds
Pi sulfur bonds
Stacked pi pi bonds
T shaped pi bonds
Pi Alkyl  bonds
Alkyl alkyl bonds
Sulfur bonds
SIX1-I-3, SIX2-I-3, SIX8-I-3 Hydrogen bonds
Pi donor bonds
Pi cation bonds
Pi anion bonds
Pi sigma bonds
Pi sulfur bonds
Stacked pi pi bonds
T shaped pi bonds
Pi Alkyl  bonds
Alkyl alkyl bonds

Table 1: Bonds formed between the docked complexes of pathogenic SIX1, SIX2, SIX8 proteins and resistant I, I-1, I-2, I-3 proteins.

The amino acids, which demonstrated better interactions in each docked complex are shown in Tables 2-4.

  SIX1-I SIX1-I-1 SIX1-I-2 SIX1-I-3
Common interacting amino acids MET1 MET1 PRO52 MET1
ALA2 PRO3 ASP53 ARG156
LEU8 VAL7 LYS54 THR159
LEU9 ASN104 LEU57 ARG160
LEU12 ARG106 TRP59 ALA161
LEU115 VAL107 ASN60 CYS162
LEU118 ASP110 ASP61 PRO163
CYS119 LYS148 MET63 ARG191
ARG122 PRO149 GLU96 GLU193
PRO186 SER150 PHE98 VAL194
VAL187 ARG152 GLU100 LYS195
CYS188 GLU153 ARG103 ASP199
ARG191 ARG154 ASP105 ILE200
GLU193 ASP155 ARG106 GLY201
VAL194 ARG156 VAL107 ILE202
LYS195 VAL158 THR136 TYR211
ASP196 THR159 ARG139 GLU213
ARG197 ARG160 THR141 TYR280
ILE200 ALA161 LYS143  
HIS203 CYS162 LYS148  
GLU205 PRO163 VAL151  
THR209 GLN166 ARG152  
TYR211 ARG179 ARG156  
ASP243 HIS181 MET227  
TYR245 VAL183 ASN232  
LYS246 THR209 TYR234  
ARG263 TYR211    
THR271 PHE235    
ARG273 TYR237    

Table 2: The interacting amino acids between the docked complex of SIX1 to I, I-1, I-2 and I-3 proteins.

  SIX2-I SIX2-I-1 SIX2-I-2 SIX2-I-3
Common interacting amino acids TRP7 PRO22 ILE16 ALA15
VAL8 ASN85 SER17 SER17
LEU10 CYS116 ALA19 PRO22
ASP29 TRP117 PRO22 ALA23
LYS48 ASP159 GLY24 GLY24
TYR56 ASN162 ASP25 ASP25
HIS58 GLY163 HIS32 ASP114
ARG60 PHE165 PHE103 CYS116
ASP78 PRO166 ARG108 TRP117
GLU80 HIS169 ASP114 MET143
LEU82 CYS171 TYR115 ARG144
LEU83 ASN173 CYS116 ASN146
ASN85 SER174 TRP117 ASP147
GLU86 ASP175 ARG118 HIS169
TRP164 ASN181 ASP119 ALA170
PHE165 HIS182 THR141 CYS171
GLN177 ARG183 SER142  
TYR179   MET143  
ASN181   PHE157  
HIS182   TYR161  
LEU185   TRP164  
VAL188   HIS169  
TYR193   CYS171  
ASP195   ASN173  
HIS196   SER174  
ARG205   ARG183  
ASN208      
SER222      
ASN223      
GLY224      
ALA226      

Table 3: The interacting amino acids between the docked complex of SIX2 to I, I-1, I-2 and I-3 proteins.

  SIX8-I SIX8-I-1 SIX8-I-2 SIX8-I-3
Common interacting amino acids CYS116 ALA17 ASP115 ALA75
LEU119 LEU18 CYS116 ASP76
TRP126 HIS43 LEU119 THR151
ARG129 CYS94 GLU120 LYS156
GLU130 ALA95 ARG179 VAL158
ASP132 LYS156 ASP181 ARG168
PRO137 VAL158 SER207 LYS169
ARG179 ARG160 MET208 ILE171
TYR184 ARG168 GLU209 ARG172
ALA206 ARG172 PRO210 LYS174
MET208 LYS174 TRP212 HIS183
GLU209 TYR184 ASN213 TYR184
PRO210 SER188 PHE214 ARG189
TRP212 ARG189 ASP215 GLN220
ASN213 PHE193 PHE224 PHE222
PRO216 MET208 PHE226 PHE224
SER217 GLU209 PRO228 PRO228
PHE222 PHE226   PRO230
PHE224 THR227   ASN231
PHE226 PRO230   ARG234
PRO228 ARG234   GLN236
      GLY237
      THR238
      ASN240
      LEU241
      ALA242

Table 4: The interacting amino acids between the docked complex of SIX to I, I-1, I-2 and I-3 proteins.

When two proteins are docked to each other, one protein can form a complex with another protein, this can steadfastly predict which amino acid residues are situated in the contact site of the protein. Diverse features of interactive sites, such as hydrophobicity, residue tendencies, size, shape, solvent accessibility, and residue pairing inclinations are calculated by examining the interactions between two proteins in a docked complex [36]. Same approach of determining the interactive amino acid residues was used in this research work to know about the other properties of proteins.

In docked complexes proteins interact with each other or numerous small molecules with a high specificity to form a complex. A comprehensive understanding of the protein-ligand interactions is thus vital to understanding biology at the molecular level. Furthermore, information of the mechanisms accountable for the protein-ligand recognition and binding likewise simplify the discovery, design, and development of drugs [37].

From Tables 2-4 it was observed that the common interacting amino acids among all the docked complexes were Leu, Met, Pro, Phe, Ala, Gly and Lys. The SIX1 protein demonstrated grater interactions with I and I-1 resistant proteins, SIX2 protein demonstrated grater interactions with I and I-2 resistant proteins, whereas the SIX8 protein represented grater interactions with I, I-1 and I-3 proteins. From the above observations, it is suggested that all the I, I-1, I-2, and I-3 resistant proteins can be used as a remedy to provide resistance against the Foc4 to prevent the banana vascular wilt disease.

Fernandes et al. performed in vitro analysis to analyze the significance of SGE1 gene expression in the Foc virulence through post-transcriptional silencing method using a double-stranded RNA hairpin. Their analysis discovered that the Foc agents were capable to spread the rhizomes and pseudostems of the inoculated banana plants [38].

Based on previous wet lab experiments conducted to confirm the Foc4 pathogenecity against banana, this in silico analysis can be further utilized in wet labs to confirm the I genes resistance against Foc.

Conclusion

In this study, the four resistant genes are revealed to use against Foc4 in banana i.e., gene, I-1, I-2, and I-3 results that Foc4 is closely related to the tomato vascular wilt pathogen Fol by the phylogenetic analysis. It is also revealed that there is a higher similarity in the genomes of Foc4 and Fol. Therefore; the I genes of tomato can also be induced in banana to provide resistance against Foc. This investigation can help the scientists to further work to develop the resistance in banana. This will eventually lead to the improvement of Fusarium wilt disease resistance in banana.

Conflict of Interest

This research work is unique and has not been submitted to any other journal. None of the authors have challenged conflicts of interest.

Funding

No funds were provided to authors from any funding source.

Acknowledgement

Authors seriously acknowledge the sustenance delivered by the Bioinformatics International research club Abbottabad for conducting this research work. The authors also thanks to other affiliations who provided beneficial information to conduct research work.

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