Journal of Plant Science

ISSN: 2573-7988

VOLUME: 3 ISSUE: 2

Page No: 159-164

Genome-wide analysis of defensin-like genes in Coffea arabica


Corresponding Author

Weihuai Wu , and Kexian Yi 

Email: weihuaiwu2002@163.com (W.W.); yikexian@126.com (K.Y.)

Affiliation

Xing Huang 1, Thomas Gbokie Jr. 2, Baohui Liu 2, Weihuai Wu 1,*, and Kexian Yi 1,*

1   Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China; hxalong@gmail.com (X.H.); weihuaiwu2002@163.com (W.W.); yikexian@126.com (K.Y.)

2   College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; gbokiejr@gmail.com (T.G.); baohuiliu2019@163.com (B.L.)

 

Citation

Xing Huang,Weihuai Wu , Kexian Yi Genome-wide analysis of defensin-like genes in Coffea arabica(2019)SDRP Journal of Plant Science 3(2) p:159-164

Abstract

Coffee leaf rust disease caused by Hemileia vastatrix, is currently a very serious problem in coffee production and resistant varieties are the most appropriate and sustainable mitigation strategy against the disease. But the knowledge for both constitutive and inducible immune system is still limited in C. arabica. In this present study, we conducted genome-wide analysis of defensin-like genes, an important peptide family of plant constitutive immunity, in C. arabica genome. Eighteen defensin-like genes were obtained by TBLASTn method and further clustered into two groups by phylogenetic analysis. Nucleotide sequence alignment revealed insertion/deletion mutations of duplicated defensin-like genes in C. arabica. Besides, structure modeling apparently indicated two types of protein structures among the 18 defensin-like genes. This study provides an overview of defensin-like genes in C. arabica and will serve as a guideline for future study.

Keywords: Coffea arabica; defensin-like genes; phylogeny; protein structure

Introduction

Coffee is a well appreciated beverage crop in the world and a cash crop of huge socioeconomic importance for many smallholder farmers in developing countries. Coffea arabica and Coffea canephora are main commercially produced species ofo coffee [1]. Leaf rust disease caused by a biotrophic fungus Hemileia vastatrix (Uredinales: Pucciniaceae) is a very serious phytosanitory problems in the production of this crop [2]. Fungicide is an efficient way to control the disease, however, associated the production cost and concerns over the adverse effects of misapplications on the quality of coffee drinks and the environment make resistant varieties being the most appropriate and sustainable management strategy against the disease [3]. A series of resistant varieties were released, including Oeiras-MG 6851 that originated from the cross Caturra Vermelho (CIFC 19/1) and Hibrido de Timor (CIFC 832/1) [4]. But the resistance has already been broken by race XXXIII of H. vastatrix [5]. Thus, it is really of great importance to study the interaction and co-evolution between coffee and H. vastatrix. Coffee-H. vastatrix rust interactions are governed by the gene-for-gene relationship [6]. To date, transcriptome and proteome methods has been used to find resistance genes [7,8]. In spite of the reported dynamics of H. vastatrix effector candidate genes (HvECs), the existing knowledge of resistance genes (SH1-SH9) is still limited [9]. The plant immune system consists of both constitutive and inducible mechanisms [10]. Defensin is an important peptide family of plant constitutive immunity [11]. Some exogenous defensin genes have been successfully transformed in crops and improved the disease resistance of transgenic lines [12-14]. Although resistance genes (SH1-SH9) has already been used for coffee breeding, there are however few reports on coffee defensin. Here, we present the genome-wide analysis of defensin-like genes in C. arabica by the released bioinformation on Phytozome. The results provide an overview for defensin-like genes in C. arabica and will serve as a guideline for future study.

Materials & Methods

2.1. Sequence retrieval

The seed sequences of conserved domain in defensin proteins were downloaded from Pfam database under the accession of PF00304 [15]. These sequences were used as query to search against C. arabica genome database from Phytozome [16] by using TBLASTN with a cut off Expected value (E-value) of 10−5 [17]. The information of these genes on scaffold position were also obtained.

2.2. Phylogenetic analysis

A neighbor-joining (NJ) phylogenetic tree was constructed for defensin proteins using the Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 [18]. Bootstrap values from 1000 trials were used for constructing the most parsimonious tree.

2.3. Intron analysis

Obtained genomic and coding sequences of defensin-like genes in C. arabica were employed for intron analysis. The exon/intron structures were analyzed using the online Gene Structure Display Server (GSDS) with coding sequences and genomic sequences [19].

2.4. Motif analysis

Amino acid sequences were aligned using Clustal X [20]. The alignment was employed for motif analysis in DNAMAN software (Lynnon Corporation, VauDREUIL-DORION QC Canada) [21].

2.5. Structure modeling

Translated protein sequences of defensin-like genes in C. arabica were used for structure modeling by PHYRE2 Protein Fold Recognition Server [22].

Results

3.1. Identification of defensin-like genes in C. arabica

After removing the redundant sequences, we obtained 18 defensin-like genes in the genome database of C. arabica line UCG-17 (Table 1). Their genomic sequence lengths were from 476-1065 base pairs (bp). The coding sequence length of these genes ranged from 225 to 321 bp, encoding peptides of 74-106 amino acids (aa). Their scaffold locations were also obtained when sequence retrieval, which indicated that 5 gene clusters, were formed.

 

3.2. Phylogenetic and intron analysis

The 18 protein sequences were clustered into 2 groups (Figure 1A). Group I contained 16 sequences, which is far more than Group II. Group I was further divided into 2 subgroups with each containing 8 sequences. Intron/exon for each defensin-like gene was analyzed according to their genomic and coding sequences (Figure 1B). All the genes contained one (1) intron, respectively. And the intron lengths were from 239-828.

https://www.siftdesk.org/articles/images/499/1.png

Figure 1. Phylogenetic analysis of defensin proteins in C. arabica (A). Bootstrap values were from 1000 trials. Intron/exon configurations of defensin-like genes in C. arabica (B). Introns and exons drawn to scale with the full encoding regions of their respective genes with boxes indicating the exons, and lines representing introns.

Table 1. Defensin-like gene family in C. arabica.

Accession

Genomic (bp)

CDS (bp)

Protein (aa)

Intron Length (bp)

Position

Strand

Scaffold574.79

1065

237

78

828

Scaffold574:831842-832906

+

Scaffold576.489

690

321

106

369

Scaffold576:3566049-3566738

+

Scaffold576.490

495

237

78

258

Scaffold576:3568764-3569258

+

Scaffold584.42

502

246

81

256

Scaffold584:412397-412898

+

Scaffold626.198

884

225

74

659

Scaffold626:2859425-2860308

-

Scaffold637.339

666

255

84

411

Scaffold637:2549770-2550435

+

Scaffold637.340

638

234

77

404

Scaffold637:2552861-2553498

+

Scaffold637.341

491

237

78

254

Scaffold637:2554488-2554978

+

Scaffold638.445

839

237

78

602

Scaffold638:5246186-5247024

+

Scaffold663.65

502

246

81

256

Scaffold663:766014-766653

+

Scaffold671.1114

693

321

106

372

Scaffold671:9167520-9168212

+

Scaffold671.1115

476

237

78

239

Scaffold671:9169969-9170444

+

Scaffold2240.6

551

312

103

239

Scaffold2240:30833-31383

-

Scaffold2240.7

693

321

106

372

Scaffold2240:33139-33831

-

Scaffold2596.106

677

246

81

431

Scaffold2596:734206-734882

+

Scaffold2651.21

895

225

74

670

Scaffold2651:351557-352451

-

Scaffold2651.23

897

225

74

672

Scaffold2651:367736-368632

-

Scaffold2651.27

895

225

74

670

Scaffold2651:402056-402950

-

3.3. Motif analysis and structure modeling

Plant defensin family contains the gamma thionin domain (PF00304), which was also observed in C. arabica defensins (Figure 2). The alignment result indicated that two gene pairs shared the same protein sequences, i.e. Scaffold671.1114/2240.7 and Scaffold2651.21/2651.23/2651.27/626.198. We further analyzed their nucleotide sequences and found that the genomic sequences of Scaffold671.1114 and Scaffold2240.7 were the same, also between Scaffold2651.21 and Scaffold2651.27. Scaffold2651.23 shared the same sequence in coding region with Scaffold2651.21 and Scaffold2651.27, but an insertion mutation in intron region (Figure 3). Scaffold626.198 had two deletion mutations in intron region and two synonymous mutation in coding region. Besides, there was frameshift mutation caused by single nucleotide deletion in Saffold2240.6 (Figure 4).

https://www.siftdesk.org/articles/images/499/2.png

Figure 2. Alignment of defensin proteins in C. arabica. Conserved cysteine residues were highlighted with red dot.

https://www.siftdesk.org/articles/images/499/3.png

Figure 3. Mutation configurations (genomic sequence) of Scaffold2651.23 and Scaffold626.198 compared with Scaffold2651.21/2651.27. The numbers represent the length of the sequence.

https://www.siftdesk.org/articles/images/499/4.png

Figure 4. Frameshift mutation caused by single nucleotide deletion in coding sequence of Scaffold2240.6. The numbers represent the length of the sequence.

We further constructed the protein structure models by PHYRE2 Protein Fold Recognition Server. All the 16 proteins in Group I matched the d1gpsa model of PHYRE2 (Figure 5A), while the 2 proteins in Group II matched the d1n4na model from PHYRE2 (Figure 5B).

https://www.siftdesk.org/articles/images/499/5.png

Figure 5. Structure models of proteins in Group I (A) and Group II (B) based on d1gpsa and d1n4na models from PHYRE2, respectively.

Discussion

In this study, we successfully identified 18 defensin-like genes in C. arabica genome, which is far less than those characterized in Arabidopsis genome [23]. Besides, a recent study revealed a similar scale of defensin-like genes in sorghum (10), maize (20), rice (12) and brachypodium (9) genomes [24]. Such a result might however not be adequate enough to provide understanding on the evolution pattern of defensin-like genes. According to phylogenetic analysis and alignment conducted, we found that two gene pairs (Scaffold671.1114/2240.7 and Scaffold2651.21/2651.27) shared the same sequence, respectively. Scaffold671.1114 and Scaffold2240.7 could very likely be allelic genes. The C. arabica genome was not assembled as high quality as C. canephora, which further emphases the difficulty for genome assembly for tetraploid [25]. More further research works are still needed to confirm the relation between Scaffold671.1114 and Scaffold2240.7, despite the assertion that Scaffold2651.21 and Scaffold2651.27 are duplicated genes. Because they are from the same scaffold, the 5 gene clusters should be also generated by whole genome duplication [26]. Duplicated genes are commonly accompanied with expansion of gene family and mutation of gene function. Our result provided an example for gene mutation at early stage (Figure 3). Besides, the frameshift mutation in Scaffold2240.6 is believed to cause the change in the tail part of the protein sequence (Figure 2; Figure 4). This mutation might cause an irreversible change of the protein function. It is worth noting that the 16 defensins categorized in Group I matched d1gpsa model in PHYRE2, and their structure models were consistent with reported defensins [11]. Moreover, Group II defensins only had partial structure compared with Group I, which might weaken their antifungal activity. Recombinant expression analysis for these defensins in C. arabica would be necessary and efficient to characterize their functions in future studies [27].

Acknowledgement

We would like to thank Margit Laimer from University of Natural Resources and Life Sciences, BOKU-VIBT (Vienna 1190, Austria) for her thorough suggestions on the experimental design


Author Contributions: Conceptualization: X.H., W.W. and K.Y.; Formal analysis: X.H.; Funding acquisition: W.W. and K.Y.; Investigation: X.H., T.G. and B.L.; Supervision: K.Y.; Writing—original draft: X.H.; Writing—review and revise: T.G.

Funding: This study was funded by National Key R&D Program of China (2018YFD0201100), FAO/IAEA Collaborative Research Project (20380), International Exchange and Cooperation Project of the Ministry of Agriculture (SYZ2019), Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (1630042017021, 1630042019030), Special Funds for Efficient Tropical Agriculture Development of Hainan Province (UF37721).

References

  1. Davis, A.P.; Tosh, J.; Ruch, N.; Fay, M.F. Growing coffee: Psilanthus (Rubiaceae) subsumed on the basis of molecular and morphological data; implications for the size, morphology, distribution and evolutionary history of Coffea. Bot. J. Linn. Soc. 2011, 167, 357-377.

    View Article           
  2. Zambolim L. Current status and management of coffee leaf rust in Brazil. Trop Plant Pathol 2016, 41, 1-8.

    View Article           
  3. Várzea, V.M.P.; Marques, D.V. Resistance, population variability of Hemileia vastatrix vs. coffee durable. In Durable resistance to coffee leaf rust; Zambolim, L.; Zambolim, E.; Várzea, V.M.P., Eds.; Publisher: UFV Press, Viçosa, Brazil, 2005; pp. 53-74.

  4. Pereira, A.A.; Zambolim, L.; Chaves, G.M.; Sakiyama, N.S. Cultivar de café resistente à Ferrugem: Oeiras-MG 6851. Rev. Ceres. 2000, 46, 121-124.

  5. Capucho, A.S.; Zambolim, E.M.; Freitas, R.L.; Haddad, F.; Caixeta, E.T.; Zambolim, L. Identification of race XXXIII of Hemileia vastatrix on Coffea arabica Catimor derivatives in Brazil. Australas Plant Dis. Notes 2012, 7, 189-191.

    View Article           
  6. Flor, H.H. Inherence of pathogenicity in Melampsora lini. Phytopathol. 1942, 32, 653-669.

  7. Guerra-Guimarães, L.; Tenente, R.; Pinheiro, C.; Chaves, I.; Silva Mdo, C.; Cardoso, F.M.; Planchon, S.; Barros, D.R.; Renaut, J.; Ricardo, C.P. Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix. Front. Plant Sci. 2015, 6, 478. PMid:26175744

    View Article      PubMed/NCBI     
  8. Florez, J.C.; Mofatto, L.S.; Freitas‑Lopes, R.L.; Ferreira, S.S.; Zambolim, E.M.; Carazzolle, M.F.; Zambolim, L.; Caixeta, E.T. High throughput transcriptome analysis of coffee reveals prehaustorial resistance in response to Hemileia vastatrix infection. Plant Mol. Biol. 2017, 95, 1-17. PMid:29094279

    View Article      PubMed/NCBI     
  9. Maia, T.; Badel, J.L.; Marin-Ramirez, G.; Rocha, C.M.; Fernandes, M.B.; Silva, J.C.F.; Azevedo-Junior, G.M.; Brommonschenkel, S.H. The Hemileia vastatrix effector HvEC6 suppresses bacterial blight symptoms in coffee genotypes with the SH1 rust resistance gene. New Phytol. 2017, 213, 1315-1329. PMid:27918080

    View Article      PubMed/NCBI     
  10. Doehlemann, G.; Hemetsberger, C. Apoplastic immunity and its suppression by filamentous plant pathogens. New Phytol. 2013, 198, 1001-1016. PMid:23594392

    View Article      PubMed/NCBI     
  11. Thomma, B.P.; Cammue, B.P.; Thevissen, K. Plant defensins. Planta 2002, 216, 193-202. PMid:12447532

    View Article      PubMed/NCBI     
  12. Swathi, A.T.; Divya, K.; Jami, S.K.; Kirti, P.B. Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep. 2008, 27, 1777-1786. PMid:18758784

    View Article      PubMed/NCBI     
  13. Ntui, V.O.; Thirukkumaran, G.; Azadi, P.; Khan, R.S.; Nakamura, I.; Mii, M. Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot. Plant Cell Rep. 2010, 29, 943-954. PMid:20552202

    View Article      PubMed/NCBI     
  14. Gaspar, Y.M.; Mckenna, J.A.; Mcginness, B.S.; Hinch, J.; Poon, S.; Connelly, A.A.; Anderson, M.A.; Heath, R.L. Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1. J. Exp. Bot. 2014, 65, 1541. PMid:24502957

    View Article      PubMed/NCBI     
  15. El-Gebali, S.; Mistry, J.; Bateman, A.; Eddy, S.R.; Luciani, A.; Potter, S.C.; Qureshi, M.; Richardson, L.J.; Salazar, G.A.; Smart, A.; et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019, 47, D427-D432. PMid:30357350

    View Article      PubMed/NCBI     
  16. Goodstein, D.M.; Shu, S.; Howson, R.; Neupane, R.; Hayes, R.D.; Fazo, J.; Mitros, T.; Dirks, W.; Hellsten, U.; Putnam, N.; et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012, 40, D1178-D1186. PMid:22110026

    View Article      PubMed/NCBI     
  17. Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389-3402. PMid:9254694

    View Article      PubMed/NCBI     
  18. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870-1874. PMid:27004904

    View Article      PubMed/NCBI     
  19. Hu, B.; Jin, J.; Guo, A.Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: an upgraded gene feature visualization server. Bioinform. 2015, 31, 1296-1297. PMid:25504850

    View Article      PubMed/NCBI     
  20. Jeanmougin, F.; Thompson, J.D.; Gouy, M.; Higgins, D.G.; Gibson, T.J. Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 1998, 23, 403-405. 01285-7

    View Article           
  21. DNAMAN-Bioinformatics Solutions. www.lynnon.com (accessed on 20-03-2019).

  22. Bennett-Lovsey, R.M.; Herbert, A.D.; Sternberg, M.J.; Kelley, L.A. Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 2008, 70, 611-625. PMid:17876813

    View Article      PubMed/NCBI     
  23. Silverstein, K.A.T.; Graham, M.A.; Paape, T.D.; VandenBosch, K.A. Genome Organization of More Than 300 Defensin-Like Genes in Arabidopsis. Plant Physiol. 2005, 138, 600-610. PMid:15955924

    View Article      PubMed/NCBI     
  24. Wu, J.; Jin, X.; Zhao, Y.; Dong, Q.; Jiang, H.; Ma, Q. Evolution of the defensin-like gene family in grass genomes. J. Genet. 2016, 95, 53. PMid:27019432

    View Article      PubMed/NCBI     
  25. Denoeud, F.; Carreteropaulet, L.; Dereeper, A.; Droc, G.; Guyot, R.; Pietrella, M.; Zheng, C.; Alberti, A.; Anthony, F.; Aprea, G.; Aury, J.M.; Bento, P.; Bernard, M. The coffee genome provides insight into the convergent evolution of caffeine biosynthesis. Sci. 2014, 345, 1181-1184. PMid:25190796

    View Article      PubMed/NCBI     
  26. Jaillon, O.; Aury, J.M.; Wincker, P. "Changing by dou-bling", the impact of whole genome duplications in the evolution of eukaryotes. C. R. Biol. 2009, 332, 241-253. PMid:19281955

    View Article      PubMed/NCBI     
  27. Picart, P.; Pirttilä, A.M.; Raventos, D.; Kristensen, H.H.; Sahl, H.G. Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity. BMC Plant Biol. 2012, 12, 180. PMid:23035776

    View Article      PubMed/NCBI     

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