Molecular Evolution and Functional Divergence of Chitinase Gene Family in Hevea brasiliensis Genome

  1. 1.  University of Florida

Abstract

Hevea brasiliensis, the largest commercial source of natural rubber, is known to be invaded by multiple fungal pathogens, affecting the global rubber production resulting in severe economic losses. Usually, chitinases form the very first line of defense against fungal pathogens in plants. However, the chitinase gene family structure remains elusive in the rubber tree genome. A complete overview of this gene family in rubber tree is presented, underscoring the gene structures, phylogeny, and conserved features of encoded proteins. Using homology-based bioinformatic approaches, it is reported that the chitinase gene family in rubber comprise of 39 members belonging to glycoside hydrolase (GH) classes. They are represented as 7 Class I [GH 19], 1 Class II [GH 19], 16 Class III [GH18], 5 Class IV [GH19] and 10 Class V [GH18] sequences as inferred from their sequence and structural features. Moreover, Class I chitinases share sequence similarity to plant lectins. Notably, the lysozyme-like domains in Class III chitinases, also known as hevamines, provide specialized machinery for resistance against pathogens. EST and transcriptome analyses indicate the exclusive expression of Class IV and V chitinases in leaves, while others are expressed in both leaves and latex. Phylogenetic and comparative genomic analyses of the sequences reveal the constant and selective evolutionary divergence of specific classes of chitinases in rubber tree, probably, to enable the tree against the fungal attacks and as adaptive mechanism for endophytic fungi. These molecular tools would provide enormous applications in management of phytopatho-interactions in rubber tree in future.

Introduction

            Chitinases (Enzyme Commission 3.2.1.14) are universal in extant life forms starting from archaea to bacteria, fungi, plants and animals and play ubiquitous roles in breaking down chitin, the second most abundant carbohydrate in nature, after cellulose. Bacteria and fungi utilize chitinases in maintaining a balance between the large amounts of carbon and nitrogen trapped in the biomass as insoluble chitin in nature. Fungi use chitinases to disrupt the existing cell walls during cell division, while insects and crustaceans use them to degrade the old cuticle, made up of chitin. Chitinases hydrolyze chitin into simple sugars, and are classified as exochitinases and endochitinases. Furthermore, they can be classified into two families, i.e., glycoside hydrolase 18 and 19, differing in their amino acid sequences, three-dimensional structures and molecular mechanisms of catalysis. Notably, family 18 chitinases have catalytic domains of triose phosphate isomerase (TIM barrel) fold with a conserved DxDxE motif and catalyze a substrate-assisted hydrolytic mechanism (Vaaje-Kolstad et al. 2004). In contrast, the family 19 chitinases are rich in α-helices and adopt single displacement catalytic mechanisms (Brameld and Goddard, 1998). They also differ by a substrate ‘retention’ and substrate ‘inversion’ mechanisms, for family 18 and 19, respectively. Family 18 chitinases are distributed in all the five lineages of life, whereas the family 19 chitinases are exclusive to Plantae.

In plants, chitinases are strongly induced when host plant cells are challenged by pathogen stress, are implicated in relationships between plant cells and fungi (e.g., mycorrhizae associations) and bacteria (e.g., legume/Rhizobium associations) and in abiotic stress responses as noted for osmotic, salt, cold, wounding and heavy metal stresses (Grover, 2012). Plant chitinases occur as diverse groups that differ in their primary structure, isoelectric point and cellular localizations and their classification has focused on the presence of several domains, i.e., a chitin-binding domain, a hinge domain and a carboxyl-terminal extension flanking the main catalytic domain (Hamel et al. 1997). Plant chitinases are divided into seven classes (I-VII) based on their primary sequences, of which classes I, II, IV, and VII belong to the family 19 glycosyl hydrolase, while classes III, V, and VI belong to family 18 glycosyl hydrolase. Class I and IV chitinases are composed of an N-terminal chitin-binding domain or module (ChBD, CtBM) and a C-terminal catalytic domain (CatD), both connected by a linker peptide of 10- 20 residues. Class II chitinases consist of a single domain corresponding to the CatD of class I. The ChBD of class I and IV chitinases is referred to as hevein domain and is grouped into carbohydrate-binding module family 18 (CBM18). Several plant lectins are composed of one or multiple-repeat hevein domain(s).

The Para rubber tree, Hevea brasiliensis, is the sole commercial source of natural rubber. However, this commercially important tree, is globally affected by many fungal diseases, resulting in declines in population, mortality and hence, rubber production. Moreover, rubber tree is known to harbor vast numbers of symbiotic fungi. Chitinases form the first line of defense against fungal pathogens. However, chitinases and their roles in rubber tree are poorly understood. With the completion of the draft genome sequence (Rahman et al. 2013), and upsurge in the amount of collected transcriptomes from bark (Li et al. 2012), leaf and latex (Xia et al. 2012), flowers, fruits, bark (Chow et al. 2014), latex (Salgado et al. 2014, Chow et al. 2014, Wei et al. 2015) of H. brasiliensis, it is expected that most of the chitinases could be identified and annotated. The genome, transcriptomes and publicly available EST sequences provide us with enormous resources with which the biological roles and specializations adopted by these enzymes, during the course of evolution could be examined. Thus, we aimed to study the gene family structure of the chitinases in the rubber genome, their structural diversities, esp. in the catalytic domains and possible evolutionary relationships and shed light on its comparative genomics aspects.

Results and Discussion

Identification and in silico characterization of chitinase gene family

The major objective of this study was to identify and characterize the entire chitinase gene family in rubber tree. With the genome sequence of rubber tree available with us and the limited amount of EST data currently available in web-based databanks, it was possible to describe the entirety of the chitinase family. One of the aims of this study is to uncover details about the entire complement of chitinases, based on their primary and secondary structure, to understand the possible functional correlations with their structure, variability in length and composition and any distinct features across the entire gene family.

Formerly, barley and Hevea chitinases have been classified as b-type (classes I, II and IV) and h-type (classes III and V) chitinases, respectively (Beintema et al. 1994). Homology based search approaches led to the identification of 78 potential chitinase candidates in the rubber tree genome. Upon re-annotation and sequence-structure-function analyses, exclusion of identical sequences, rejection of partial and fragmented sequences, we finally identified 39 chitinases, i.e., 7 Class I [GH 19], 1 Class II [GH 19], 16 Class III [GH18], 5 Class IV [GH19] and 10 Class V [GH18], which were renamed according to the Class to which they belong, followed by a serial number [Figure 1]. In fact, another model plant from the same order Malpighiales as rubber tree, Populus trichocarpa was demonstrated to consist of 37 chitinases (Jiang et al. 2013), which are similar to what found here. The class I chitinases, show higher similarity to plant lectins. Presence of a single class II chitinase is rather interesting. Thus, rubber tree has 41% of chitinases belonging to Class III, which are ‘hevamines’, i.e., enzymes with both lysozyme and chitinase activities. Class IV chitinases show very high sequence similarity to the class I chitinases [Supplementary Figures 1-5]. The latex of H. brasiliensis is known to contain a very high level of chitinases, i.e., up to 25 % of the lutoid-rich soluble protein fraction from a latex preparation was chitinolytic (Martin, 1991), mostly, identified as hevamines (Subroto et al. 1996).

Description: 94ac35a2-ad4a-48d9-8d53-5fec29b08ac9-1 (2)

Figure 1. Chitinase gene family architecture in rubber tree genome. 

Chitinases are represented by five major classes in rubber tree, i.e., I, II (1-membered), III, IV and V. Distribution of signal peptide, transmembrane domains, Prosite motifs as well as patterns are shown.

Multiple sequence alignment and sequence features

The phylogenetic relationship of the chitinases were examined by the multiple sequence alignments, based on their grouping of different Pfam domains, Prosite-motifs and 3D- structural features, inferred from homology-based models [Supplementary Tables 1- 2]. The chitinase genes and proteins of rubber tree fall into all known angiosperm and dicotyledonous chitinase classes, i.e., I, II, III, IV, V; while we could not find any class VI and VII chitinases in the draft genome sequence. Class III chitinases, belonging to glycosylhydrolase 18 (GH18) families, are with the largest number of members. In contrast, the genome of Arabidopsis thaliana encodes single-membered class I (At3g12500) and class III (At5g24090) chitinases (Passarinho and de Vries, 2002). Ortholog mapping against A. thaliana gene models reveal similar results [Supplementary Table 6].

The class I chitinases of rubber, show many conserved features toward the N terminal end of the proteins, i.e., MLKXR, GFYTYD, AFISAA, FGTTGD, TSH; and span a stretch of 54 amino acids. The class III shows the conserved motifs, LWNNNFLGG, LDGIDFDIE, and WDDLARXLS, spanning 51 amino acids. Class IV chitinases show the motifs located towards the C terminal of the proteins, DPXXSFKXALW, GXGXTIRAXN, and NECXGGXYYTDYCXQXGV, spanning a region of 42 amino acids. The class V chitinases show the mid-sequence conserved feature, DG (I/L) D (I/L) D(Y/W) EH. Also, The chitinase 19_1 signature C-x(4,5)-F-Y-[ST]-x(3)-[FY]-[LIVMF]-x-A-x(3)-[YF]-x(2)-F-[GSA] and 19_2 signature [LIVM]-[GSA]-F-x-[STAG](2)-[LIVMFY]-W-[FY]-W-[LIVM] are localized in all GH 19 chitinases [Figure 2]. Sequence comparisons, conserved motifs showed that the rubber tree chitinases can be clearly classified into 4 large groups, i.e., classes I, III, IV, V and the single-membered class II.

Description: 2

Figure 2. Sequence logos for the 4 classes of chitinases in rubber tree.

a. Class I, b. Class III, c. Class IV, d. Class V.

Class I and IV share, the Prosite motif ‘CHIT_BIND_I_1’ (PS00026) and ‘CHITINASE_19_1’ (PS00773) and ‘CHITINASE_19_2’ (PS00774), as well as Pfam domains for Chitinase class I (‘Chitin_bind_1’, PF00182) and Chitin recognition protein, (‘Glyco_hydro_19’, PF00187, CDD: 29557) and CDD: cl00045, ChtBD1 superfamily, while, for some sequences an organellar (i.e., vacuolar) localization signal in the form of signal peptide is discernible. The chitin binding domain, ChtBD1(CDD:28917), is involved in recognition or binding of chitin subunits, carry a fold analogous to hevein and occurs in plant and fungal proteins that bind N-acetyl glucosamine, plant endochitinases, wound-induced proteins. Class III chitinases show the presence of glycosylhydrolase family 18 characteristic Prosite motif ‘CHITINASE_18’ (PS01095), Glycosyl hydrolases family 18, specific Pfam domain: ‘Glyco_hydro_18’, PF00704 and CDD: cl10447, GH18_chitinase-like superfamily and CDD: cl00222 lysozyme_like superfamily signatures. Class V chitinases also show the presence of Pfam domain: ‘Glyco_hydro_18’, PF00704 [Supplementary Figure 6]. It is noteworthy, that members of class I, III and IV, all GH 19 chitinases, share structural similarity to several lysozymes, though the sequence similarity is lower.

Of the various, motifs related to post-translational modification of the chitinases, it is observed that, the N-glycosylation motifs (PS00001) N-{P}-[ST]-{P}) are absent in class I, scanty in class III, IV and densely distributed in some class V chitinases. The amidation site motif (PS00009, x-G-[RK]-[RK]) are absent in class V chitinases, whereas, the protein kinase C phosphorylation site (PS00005, [ST]-X-[RK]) is seen distributed randomly over all classes of proteins. Cysteine-residues, forming disulphide bridges, are densely distributed in the class I and IV chitinases, i.e., 8-18 sites; more specifically towards the N-terminal end of sequences. On the other hand, class III and V chitinases show moderate and sparsely distributed cysteine residues, respectively [Supplementary Figure 5].

Phylogenetic analyses and evolutionary significance of the gene family

When a maximum likelihood (ML) tree was constructed taking into account the 39 rubber tree and five representative chitinase orthologs from A. thaliana, the Chitinases were clearly separated into five classes [Figure 3]. Furthermore, the ML-tree also shows, that class I and IV chitinases closely related, i.e., presence of Prosite motif PDOC00839 (FDGVDIDWE) containing the crucial glutamate acting as H-donor in the chitinolytic process. Similarly, class III and V chitinases are closely related. In fact, in the phylogenetic tree it is clearly evident that the A. thaliana genes are clustered with the rubber tree chitinases in a class specific manner, there by indicating the absence of concerted evolution. To investigate the involvement of Darwinian positive selection in divergence of chitinase gene family after duplication, the substitution rate ratios of nonsynonymous (dN or Ka) versus synonymous (dS or Ks) mutations (dN/dS or Ka/Ks) were calculated for 17 pairs of paralogues. It is well known that Ka/Ks= 1 suggests pseudogenization with neutral selection, Ks/Ks < 1 is negative or purifying selection and Ka/Ks >1 shows positive selection. In this study, a majority of the pairs showed a Ka/Ks < 0.5 (i.e., 0177- 0.469), while in just one case, i.e., Class_I_3- Class_I_4 pair it was >1, indicating a positive selection [Supplementary Table 11]. Moreover, plant chitinases and lysozymes are likely to have arisen from one coancestor by divergent evolution (Monzingo et al. 1996), while class I and class II chitinase genes evolved from the same ancestral gene and a basic class II chitinase is a putative ancestor of basic class I and acidic class II chitinase genes (OhmeTakagi et al. 1998). Moreover, the class IV chitinases, which are phylogenetically connected with class I and II chitinases (Hamel et al. 1997; Gomez et al. 2002), are thought to have evolved from a class I chitinase gene by four deletions in the coding sequence (Araki and Torikata, 1995).

Description: 8106d131-a48e-49a3-a9cf-f04587912579-3

Figure 3. Molecular Phylogenetic analysis by Maximum Likelihood method indicative of the organization of the rubber tree chitinase gene family.

The evolutionary history was inferred by using the Maximum Likelihood method based on the Poisson correction model. The bootstrap consensus tree inferred from 1000 replicates and is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 80% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 44 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 624 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2007). Included in the analyses were representative chitinases from Arabidopsis thaliana belonging to classes I to V.

Class I chitinases

The 7-membered class I chitinases of rubber tree are 14.87 (145 aa) to 34.71 kDa (321 aa) in size, with a theoretical pI range of 5.05 to 7.98, a highly negative GRAVY (-0.059 to -0.483), low aliphatic index (50.84 – 64) and large number of cysteine residues (8-19). Presence of vacuolar targeting signal peptides in four members indicates their involvement in secretory pathways. A class I chitinase from latex, known as Hev b 11, carries a N-terminal domain highly homologous to hevein (Hev b 6.02) and shows 70% similarity to basic endochitinase from avocado, was isolated from latex (O'Riordain et al. 2002). All class I chitinases characterized show IgE-binding capacity like hevein, hydroxylation of proline residues and are implicated in type-I allergy from natural rubber latex (NRL) proteins. However, the latex allergenic proteins Hev b 1 and Hev b 3 do possess the IgE-binding or chitinolytic activities, but are involved in rubber biosynthesis as rubber elongation factor (REF) and small rubber particle protein (SRPP) (Wagner et al. 1999), respectively. Class I chitinases are predominantly expressed in roots, flowers and leaves and are not induced upon wounding (Samac et al. 1990). However, studies show that ethylene-dependence and pathogen-specificity are major events leading to the induction of class I chitinases (Verburg and Huynh, 1991, Thomma et al. 1999). Class I chitinases exhibit three to five times higher antifungal activity than their catalytic domains alone and class II chitinases, and their antifungal properties have been well established (Suarez et al. 2001, Truong et al. 2009). Understandably, constitutive expression of the rice class I chitinase, OsChia1b, in grapevine, Italian ryegrass and many commercial crops have demonstrated enhanced resistance to fungal diseases (Yamamoto et al. 2000, Takahashi et al. 2005). Similarly, Class I secretory endochitinase, McCHIT1, from bitter melon was demonstrated to enhance resistance against Phytophthora nicotianae, Verticillium, Magnaporthe grisea (rice blast) and Rhizoctonia solani (sheath blight), when heterologously expressed as transgenes (Li et al. 2009). Class I Chitinase, Mhchit1, from Malus hupehensis showed elevation in expression levels, upon treatment with salicylic acid, ethylene, methyl jasmonate, 1-aminocyclopropane-1-carboxylic acid and showed enhanced resistance to apple aphid Aphis citricota and fungus Botrytis cinerea as a transgene (Zhang et al. 2011). Similarly, a tall fescue turf grass class I chitinase, FaChit1 was shown to be activated by fungal elicitors, dehydration, ethylene, and mechanical wounding (Wang et al. 2009). These evidences clearly demonstrate the critical role conferred by class I chitinases in phyto-patho interactions and hence biotechnological intervention of rubber tree clones with class I chitinases would possibly produce resistant varieties that are responsive to hormonal treatments.

Class II chitinases

The sole class II chitinase of rubber tree identified is a 30.62 kDa (276 aa) protein, with a basic theoretical pI of 8.31, a negative GRAVY (-0.279), low aliphatic index (66.88), with 9 cysteine residues and is predicted to be tonoplastic. Class II chitinases lack both the N-terminal cysteine-rich region and the C-terminal extensions, whereas they have high sequence and structural similarity to that of class I chitinases. Class II chitinases are known to be responsive to treatment with fungal spores as well to ethylene and salicylic acid (Kellmann et al. 1996). Interestingly, they are not only involved in plant defense, but also constitutively expressed in different organs and have multiple distinct roles in endogenous regulatory functions in development (Ponath et al. 2000). In A. thaliana, AtCTL1 belongs to the class II family of chitinases that lack a Cys-rich lectin domain in the N-terminal region of the protein that has a role in chitin binding, is non-enzymatic, but plays a roles in primary root growth, development and architecture (Hermans et al. 2010). A class II chitinase is accumulated in the bark tissues in response to cold hardiness of stems in high bush blueberry, Vaccinium corymbosum L. (Kikuchi and Masuda, 2009). Furthermore, transgenic wheat plants constitutively expressing a barley class II chitinase conferred resistance against the powdery mildew and leaf rust pathogens (Oldach et al. 2001). These evidences suggest that the single class II chitinase could be critical in determining improved resistance against fungal attacks, when over expressed.

Class III chitinases

Class III chitinases, also known as ‘hevamines’, from rubber tree were known as early as 1976 (Archer, 1976). Besides, the first class III chitinase (29-30 kDa) was crystallized (Scheltinga et al. 1996) and was found to be homologous to family 8 of pathogenesis related protein (PR-8). They vary in size from 5.4 (459 aa) to 16.3 kDa (149 aa), and their theoretical pI ranges are both acidic (4.49 to 6.49) to basic (9.82 to 8.09). The GRAVY values are high (-0.288 to 0.04) compared to other classes of chitinases, have higher aliphatic index (70.4- 92.4) and are moderate in the number of cysteine residues. Hevamines were thought to possess both lysozyme and chitinase activities (Rozeboom et al. 1990). The class III chitinases in rubber tree consists of a universal catalytic site with the residues, ‘DFDIE’. However, later on, upon further biochemical characterization, it was shown that, hevamines cleave peptidoglycans, thus qualifying as peptidoglycan cleaving chitinases, like the family 18 glycosylhydrolases, rather than the glycosidic bond cleaving lysozymes (Bokma et al. 1997). However, later on, the lysozyme activities in latex products were attributed to separate lysozymes (27 kDa, pI 9.5), involved in defense responses as well as in type-I allergenic responses (Yagami et al. 1995). Furthermore, previous studies show that the active sites in hevamines carries the residues Asp125, Glu127, and Tyr183, involved in interacting with the -1 sugar residue of its substrates and mutants showed the critical roles of Asp and Tyr residues in enzyme catalysis rate and mechanisms (Bokma et al. 2002). Enzyme kinetics studies using substrates such as hexa-N-acetyl glucosamine and inhibitors like allosamidin, it was concluded that hevamines have at least six sugar binding sites in the active site (Bokma et al. 2000). The conserved domain family “G18_hevamine_XipI_class_III” includes xylanase inhibitor Xip-I, and the class III plant chitinases such as hevamines, concanavalin B, and PPL2, all of which have a glycosyl hydrolase family 18 (GH18) domains. Presence of signal peptides in 11 out of the 16 members indicates their involvement in vacuolar roles during defense responsiveness. Class III chitinases are developmentally regulated as well as induced by pathogens (Samac and Shah, 1991). In soybean, a class III acidic endochitinase was shown to be involved defense, development and dormancy events in seeds (Yeboah et al. 1998). In Lotus sps, a class III chitinases, Ltchi7, expressed only in nodules and roots, was shown to be drought stress responsive, as well as induced by salt stress, hydrogen peroxide and abscisic acid (Tapia et al, 2011). A class III chitinase from pomegranate seeds, PSC, was shown to be involved in calcium storage in amyloplasts, a non-defensive function (Lv et al. 2011). Moreover, class III chitinases from H. brasiliensis also consisted of the conserved catalytic domain sequences of family 18 glycoside hydrolases (Karlsson and Stenlid, 2009) e.g., LDGIDFDIE. Presence of six cysteine residues at conserved positions (Kim et al. 1999) and a N-terminal signal peptide that direct the matured protein to the apoplasm (Bendtsen et al. 2004) and the primary structure suggested a strong homology to class III chitinases (Collinge et al. 1993). Response to multiple physiological events in a plant’s life cycle, particularly the abiotic stress responsiveness and expansion in numbers indicate that the hevamines have diverged alongside evolution to bestow molecular defense tools to its defense armory.

Class IV chitinases

The class IV chitinases resemble class I chitinases in structural features, but are considerably smaller due to a series of four deletions distributed along the N-terminal chitin-binding domain, a glycine-rich hinge region and the catalytic domain [Supplementary Figure 7]. Class IV chitinases have 7 out of 8 cysteines position common with class I type, but are shorter than class I chitinases. Thus, the catalytic cleft is shorter and wider than that of class I and II chitinases (Ubhayasekera et al. 2009). Rubber tree class IV chitinases range in size from 33.6 to 23.8 kDa, with acidic theoretical pI ranging from 4.15 to 6.03, a negative GRAVY values (-0.113 to -0.68), a moderate aliphatic index (58 to 72.2) with relatively high cysteine residue counts (5-16). Presence of signal peptides in 3 out of the 5 members indicates their localization to the secretory pathways in the cell. In fact, it is well established that, like the class II chitinases, class IV chitinases are secreted to the apoplast, lack the C-terminal extension found in most class I chitinases (Graham and Sticklen, 1994) and do not share homology with class III and V chitinases. A conserved motif (N-Y/F-NYG) is present in class IV chitinases just two amino acids after the antigenic region, and is essential for hydrolytic activity (Verburg et al. 1993). Potential N- linked glycosylation sites (N-X-S) are present at the C-terminal ends. Despite their phylogenetic and sequence differences, both class II and class IV chitinases have a common catalytic mechanism, as evidenced by the fact that they both contain the putative key catalytic residues in the main catalytic domain (Mitsunaga et al. 2004). The class IV chitinases are induced upon fungal infection and play major roles in plant defense system (Liliane et al. 1997), respond to a broader range of stress sources like virus infection, heavy metals and UV-irradiation (Margis-Pinheiro et al. 1993). They are also known to be involved in embryo development (de Jong et al. 1993) and a variety of ethylene- induced processes such as senescence (Hanfrey et al. 1996) and ripening (Robinson et al. 1997).

Class V chitinases

            The class V plant chitinases have a glycosyl hydrolase family 18 (GH18) domain, but lack the chitin-binding domain present in other GH18 enzymes. The GH18 domain of the class V chitinases has ‘random’ endochitinase activity in some cases and no catalytic activities. The class IV chitinases in rubber tree carry the catalytic site residues ‘DIDYE’ or ‘DLDWE’. Rubber tree class V chitinases range in size from 48.5 (439 aa) to 29.87 kDa (267 aa), with an acidic theoretical pI (4.21- 6.51, excluding one basic member with pI of 8.05), highly negative GRAVY values (-0.012 to -0.276), a higher aliphatic index (69.28- 86.14) with relatively low cysteine residue counts (1-3). Presence of signal peptides in 8 out of the 10 members indicates their involvement in vacuolar roles during defense responsiveness. Interesting to note that, one of the sequences is targeted to ‘chloroplast’.

            Class V chitinases adopt a (β/α)8 fold with a small insertion domain composed of a α-helix and a five stranded β-sheet. The longer chitinases are thought to contain six sugar binding sites, labeled as -3 (non-reducing end), -2, -1, +1, +2, and +3 (reducing end), to accommodate binding of a chitohexose, i.e., (GalNAc)6 with cleavage occurring between -1 and +1. In plants, class V chitinases share the consensus sequence DXDXE found in class III, but otherwise show very low homology to class III enzymes and there are limited reports on the enzymatic and biochemical characterization, i.e., in A. thaliana (NCBI accession no. NP_193716), tobacco (NCBI accession no. CAA54374, Melchers et al. 1994) and recently, in a cycad (CrChi-A, NCBI accession no. BAD98525, Taira et al. 2009). The class V chitinases are well characterized in A. thaliana and Nicotiana tabacum, which share all structural and functional properties, are known to be induced by the plant stress-related hormones abscisic acid, jasmonic acid and flagellin, NaCl and osmosis (Ohnuma et al. 2011). Class V chitinases from A. thaliana, are known to be differentially localized to various organelles and very little in known about their functional aspects (Passarinho and de Vries, 2002). Class V chitinases are expressed in the roots of Medicago truncatula nodulated with rhizobia (Salzer et al. 2000) and sequences with up to 50 % similarity to class V chitinases, in the form of lectins were identified in black locust (Robinia pseudoacacia) bark (Van Damme et al. 2006).

Structural features and properties

The seven class I chitinases are mostly acidic in pI (5.2-5.8), except two members, that are basic (~8), and are sized at 14.8- 34.7 kDa. Ten out of the sixteen of the class III chitinases, i.e., hevamines are highly basic pI (8.2-9.5), while a few are acidic (pI, 4.7-6.1); and they are sized between 16.3 to 50.4 kDa. Class IV chitinases are acidic in their pI (4.4-5.7) and shorter in size (23.8- 33.6 kDa). The pI of Class V chitinases ranged from 4.4-8.1, while their sizes are predicted range between 25.9 to 48.5 kDa. Twenty seven out of the thirty nine sequences carried an N- terminal signal peptide. Distribution pattern of amino acids reveal that class III and V chitinases, are rich in isoleucine, leucine and serine residues, while class V chitinases are rich in asparagine, glutamate, phenylalanine, histidine, lysine, methionine and valine residues. Class V are rich in polar residues, while class I contain higher number of non-polar residues and vice versa. Recently, it was shown that a triad of polar residues is implicated in pH specificity of the acidic mammalian chitinase (AMCase) activity (Olland et al. 2009). Class V chitinases also have higher number of charged residues, both acidic and basic. In contrast, class III chitinases are lowest in terms of basic and charged residues. Similarly, Class I and IV chitinases are high in cysteine residues, while class V chitinases have the lowest number of cysteines [Supplementary Table 10]. Conserved positions of Cys residues were proposed as evidence or identically located disulfide bridges or cysteine residues towards classification of chitinases (Beintema et al. 1994). Protein structures evolve more slowly than their sequences. Structure-based multiple sequence alignment methods are expected to be more accurate than sequence-only-based multiple sequence alignment methods. All the 39 rubber tree chitinases were submitted to the automated comparative protein modeling server (http://www.expasy.org/swissmod/SWISS-MODEL.html). Proteins with the highest identity in amino acid sequence were chosen as the modeling templates and were proceeded with. Results suggest, that all most all of the class III chitinases shared homology with the PDB entry, 2hvmA (hevamine from H. brasiliensis), the class I with 3cqlB (chitinase from Carica papaya), class IV with 3hbeX (chitinase from Picea abies) and class V with 3simB (GH 18 chitinase from Crocus vernus) [Supplementary Table 4]. All the class III chitinases show the (β/α) 8 TIM barrel structure [Supplementary Figure 5].

For all family 18 chitinases, the aromatic residues in the catalytic sites, specifically, the tyrosine residues seems to be nicely conserved, indicating it’s essential role in substrate-assisted catalysis (Watanabe et al. 2003). Previous studies on Chitinase A from Serratia marcescens (ChiA, 1ffq), Chitinase B from S. marcescens (ChiB, 1e6r), Chitinase 1 from Coccidioides immitis (Chi1, 1ll4) and hevamine from H. brasiliensis (Hev, 1llo) reveals that the interactions made by the three aromatic catalytic residues influencing the active site are unique (Olland et al. 2009). In rubber tree, the class II chitinases, share a rather simple topology; while the class I and IV and class III and V chitinases, show structural and sequence similarities [Figure 4].

Description: 4

Serial No.

Chitinase Class Number

Gene Model

Closest neighbouring Gene (Upstream/ Downstream)

1

Class_V_6

HBM_contig302151_s132607c3_gene2

Laccase

2

Class_V_5

HBM_contig322565_s141866c3_gene1

Laccase, Protein Kinase

3

Class_V_4

HBM_contig1329337_s432362c4_gene2

NAC domain-containing protein

4

Class_III_5

HBM_contig1321967_s426707c2_gene2

Protein kinase

5

Class_III_4

HBM_contig1310278_s418802c3_gene1

Protein kinase

6

Class_III_16

HBM_contig542487_s252517c4_gene2

Pentatricopeptide repeat-containing protein; WRKY transcription factor

7

Class_III_1

HBM_contig350423_s154843c12_gene1

Protein kinase

8

Class_II_1

HBM_contig317814_s139612c6_gene2

WRKY transcription factor

9

Class_I_2

HBM_contig376395_s167879c1_gene1

Pentatricopeptide repeat-containing protein

10

Class_I_1

HBM_contig412336_s186887c8_gene1

NAC domain-containing protein

Figure 4. Structure and topology of class I-V chitinases in rubber tree. 

a. Topology of chitinases of class’s I-V showing N- and C- terminal ends alpha-helices (red rods) and beta-sheets (magenta arrows); b. 3-D homology models of typical representative rubber tree chitinases of class’s I-V.

Genome-wide distribution of rubber tree chitinases

Interestingly, an attempt to localize the 39 chitinases, to the rubber genome sequence at the level of contigs, scaffolds and super scaffolds, revealed that they could not be grouped or assigned to closely placed loci. However, their involvement in defense responses through a wide-ranging mechanism was evident from the neighborhood gene information, either up-stream or down-stream, in the scaffolds. It was found out that, the class V chitinases are in close vicinity to ‘laccases’, class III to ‘protein kinases’. On the other hand, the chitinases from class I, II, III and IV, were closely placed in the same genomic stretch, with ‘pentatricopeptide repeat-containing (PPR) protein’, ‘WRKY transcription factors’, ‘NAC domain-containing proteins’, in addition to the earlier mentioned genes [Table 1].

Table 1.  Neighboring genes of chitinases in rubber tree genome. Gene models located both upstream and downstream of all the chitinases were annotated to find the closest neighboring gene involved in disease resistance.

 

Structural evolution of rubber tree chitinase gene family

At nucleotide levels, the chitinase genes show an average GC content of 44.3 %. However, majority of the class I and IV chitinase genes are higher in GC content (i.e., 46-48 %); while class V chitinases are lower in GC content (i.e., 38-41%) [Supplementary Table 10]. The GRAVY (Grand Average of Hydropathy) score of chitinases are largely negative, thereby indicating a hydrophilic propensity. The chitinase genes in each separate class share a higher degree of identity with each other than with any other genes in the genome. They are also less similar to any chitinase genes belonging to the Euphorbiaceae, indicating they are results of tandem duplications and possible functional divergence. The results from a PSI-BLAST indicate, that the rubber tree chitinases share strong sequence similarities, without any particular taxonomical relevance i.e., class I with chitinases from Oryza sativa (monocot), Vigna unguiculata, Leucaena leucocephala (legumes) and Vitis vinifera (ancestral eudicot stage); class III with Panax ginseng (an asterid), Malus domestica (a fabid), Ficus pumila (a rosid); class IV with the Phaseolus vulgaris, M. truncatula (legumes) symbiosis- type chitinases, while the class V share with chitinases from Cycas (gymnosperm), Pteris ryukyuensis (pteridophyte), tulip (liliopsid monocot) [Supplementary Table 3]. Loss and gain of introns are also evident in the rubber tree chitinase gene family. While, the class I chitinases have either none or a small number (1- 3) of introns, the class II has one intron, class III has one to three introns, class IV have mostly a single intron, while class V have invariably only one intron. A vast majority of are intron-less [Supplementary Figure 8]. This investigation, also re-affirms that a few hevamines (class III chitinases) reported earlier may not possess introns (Bokma et al. 2001).

Physiological role of chitinases as allergens and in rubber production

            Natural rubber latex (NRL) is associated to hypersensitivity, and the allergen cross-reactivity is due to IgE antibodies that recognize structurally similar epitopes on different proteins that are closely related. They are also implicated in food allergy (Volpicella et al. 2014). This allergenicity is associated with the ‘hevein’ domain present in class I chitinases, heveins and several other allergenic proteins across plant kingdom i.e., in avocado, banana, chestnut, kiwi, peach, tomato, potato and bell pepper. In fact, we reaffirmed that all the 7 class I chitinases of rubber tree, containing the type-1 chitin-binding domain are allergenic. They tend to poses the experimentally determined IgE motif SQ(W/Y/F)GWC, invariably; while 4 of the class five class IV chitinases are potential allergens as well [Supplementary Table 8].

            In addition, chitinases show several fold expression levels in laticifers than in leaves (Kush et al. 1990). In the transcriptome obtained from leaves (Rahman et al. 2013), we observed the expression of class I chitinases 3 and 7 (EST as well); class III chitinases 9 (EST) and 13 (EST as well), class IV chitinases 1 and 3 and class V chitinases 2, 9 and 10. In fact, higher transcript levels of a chitinase isoform has been implicated in ethylene induced delay in coagulation of latex flow, a clonal characteristic, there by nullifying the coagulating effects of hevein and its binding receptor (Chrestin et al. 1997). The expression levels of class III chitinases, ‘hevamines’(GenBank Accessions AJ010397, CAA09110), both carrying vacuolar targeting signal peptides, were up-regulated 212- folds within 15 minutes of mechanical wounding, while upon exogenous application of methyl jasmonate (MeJA) the chitinase clone is down-regulated within 1 hour of trigger (Duan et al. 2010). Besides, chitinase and glutamine synthetase activities have been shown to be specifically modulated by ethylene in the rubber tree (Coupe and Chrestin, 1989). It is also established that, hevein, a lectin- like protein is involved in the coagulation of latex, mediated by ethylene (Gidrol et al. 1994). It is possible that, ethylene has a role to play in developmental regulation of chitinase genes (Passarinho and de Vries, 2002). Ethylene (i.e., commercially well known as ‘ethephon’) is known to stimulate and increase the latex yield in rubber trees (Coupe and Chrestin, 1989; Pujade-Renaud et al. 1994). Recently, comparative proteomics approaches helped unveil that several of the main lutoid proteins, such as glucanase, hevamine, and hevein, that belonged to chitinases and glucanases, appeared to play crucial synergistic roles in rubber particle aggregation process (Wang et al. 2013). In fact, endochitinase and β-N-acetylglucosaminidase (NAGase) are abundant in rubber latex serum (Sukprasirt and Wititsuwannakul, 2014). All these evidences strongly implicate major roles of chitinases in rubber biosynthetic process and latex production.

Functional divergence in chitinases: armory in defense against fungi and adaptation to endophytes?

            To provide a broad survey of rubber tree chitinase gene expression, ESTs and cDNA from transcriptomes were analyzed in terms of tissue of origin in the annotated databanks and compared. In silico gene expression studies indicate that, the class I and III chitinases are expressed both in leaves and latex; whereas we did not find any expression of class IV and V chitinases in latex producing laticifer tissues [Supplementary Table 9]. The foliage and the sapwood of rubber tree, are known to harbour an enormous trevor of endophytic fungi (Gazis and Chaverri, 2010; Rocha et al. 2011). Thus, it is likely that, the diverse arsenal of chitinases, are expressed differentially for a symbiotic endophyte or a pathogen. Moreover, the number of endophytes identified outgrows the number of phytopathogens by a huge number in rubber tree (Rocha et al. 2011). Hence, it is not surprising that the genome boasts more class V chitinases than the class IV counterparts. In fact, in M. truncatula, it was clearly demonstrated, that the expression patterns of symbiosis-specific class V and defense-related class IV chitinases varied greatly depending on the cues presented to the plant (Salzer et al. 2004). Furthermore, specialization in function, among a few members of a particular class of chitinase is known. In M. truncatula, it was observed that two class III chitinases, expressed exclusively after the establishment of arbuscular mycorrhiza, but never during interactions with pathogens and rhizobia, where class I, II and IV chitinase genes were strongly expressed (Salzer et al. 2000). Moreover, in rubber tree, the basic vacuolar class III chitinases is implicated with roles in plugging of the latex vessels by coagulating negatively charged rubber particles, attributed to a process of charge neutralization (Subroto et al. 1996) and hence resulting in cessation of latex flow (Jekel et al. 1991). These hevamines in rubber tree have also been attributed to a role in defense owing to the lysozyme-like activities (Martin, 1991). Thus, the 16-memebered class III chitinases, might confer differential protection and physiological advantages to the rubber during its course of life cycle, e.g. in adaptation to endophytic fungi and harsh fungal attacks. Expression of class III ESTs and transcripts in both leaf and latex indicate their diverse role in rubber tree physiology.

            Gene Ontology analyses conducted at WEGO, suggests that, they are largely involved in ‘biological process [GO: 0008150]’; ‘molecular function [GO: 0003674]’ and ‘catalytic activity GO: 0003824][Supplementary Table 7]. Many chitinases are associated with ‘hydrolase activity [GO: 0016787]’, ‘intracellular [GO: 0005622]’, ‘cellular process [GO: 0009987]’, ‘response to stress [GO: 0006950]’ etc, thereby indicating the significant role they play in defence and stress responses and the mode of action being through a hydrolytic catalysis mechanism in cell [Figure 5]. KOG classification analyses suggest that the class I, II and IV [KOG4742 (=COG3979)] III [KOG4701], and V [KOG2806 (=COG3325)] chitinases have gained functional uniqueness over the course of evolution [Supplementary Table 2].

Description: 5

Figure 5. Histogram of GO classifications of all 39 chitinases from H. brasiliensis, as inferred from WEGO analysis, based on their IPR scan hits.

Results are displayed in three main GO categories: biological process, cellular component and molecular function.

Comparative Genomics of Chitinases in Plant Kingdom

            We attempt to observe the sequence –based relationships sharing among chitinases encoded within rubber tree as well as with other genomes. It was clearly observed that the class I and IV chitinases showed very strong similarity among each other [Figure 6 a]. Similarly, the class III and V chitinases of rubber tree, showed sequence similarities to one another though limited [Figure 6 b]. To analyze the sequence similarities with other known plant chitinases belonging to class VI and VII [6 members could be correctly annotated from databases], we found out that they showed high similarities specifically to the class I, III and IV chitinases, in rubber tree [Figure 6 c]. In order, to compare the chitinase proteome across Spermatophyte sequences available in UniProt database, we observed that all the rubber tree chitinases had at least one ortholog across several species [Figure 6 d]. Interestingly, when all the annotated lectins from UniProt database consisting of Spermatophytes were pooled, it was observed that, the class I chitinases of rubber tree showed highest and exclusive similarity to lectins [Figure 6 e]. In fact, it is well know, that lectins belong to the same superfamily of proteins as chitinases, except that they differ in the loss of the enzymatic activities in the course of evolution. When the rubber tree chitinases were compared against, chitinases available from other plants, it was observed that they shared stronger sequence homology and representativeness to Medicago, Vitis and Oryza. In contrast to Oryza, the rubber tree chitinases did not show good matches with Zea chitinases [Figure 6 f-k].

Figure 6. Visualization of BLAST-based relationships and similarities among rubber tree chitinase classes and with other plant species. 

a. Class I chitinases and Class IV chitinases in rubber tree, showing very high similarity among each other [E-value cut-off, 1e-20], except one class I member. b. Class III and Class V chitinases showing sequence similarities [E-value cut-off, 1e-10]; c. Plant Class VI and Class VII chitinases (6 members), not represented in rubber tree, but showing stronger sequence similarities to Class I, III and IV chitinases in rubber tree; d. Similarities between chitinase sequences available from UniProt DB and the 39 rubber tree chitinases, reveal that all of them have a ortholog in at least one spermatophyte; e. Sequence similarity comparison between 39 rubber tree chitinases and ‘lectins’ from UniProt DB, where the Class I chitinases from rubber tree show exclusively matches with plant lectins. The 39 Chitinases from rubber tree are compared against chitinases from f. Medicago truncatulag. Vitis viniferah. Oryza sativai. Zea maysj. Ricinus communis and k. Arabidopsis thaliana.

            When the type and numbers of chitinases across various sequenced plant genomes were analyzed, using information available with GreenPhyl database, it was observed that rubber tree genome encoded a higher number of GH-18 type chitinases, i.e., 26, next only to G. max genome. In contrast, the number of GH-19 type chitinases is only 12, compared to 45 in Selaginella moelendorfii and 24 in V. vinifera genomes, respectively. However, it is noteworthy, that the ratio of GH18 to GH19 type chitinases is remarkably different in rubber tree genome, than any other genomes, i.e., 2.1, comparable only to observed values for G. max, C. papaya and O. sativa. While for other genomes the GH19 to GH18 ratio is higher [Table 2]. Recently, two chitinase-like proteins of the GH19 family from the rubber tree (HbCLP1 and HbCLP2) were cloned, expressed, and characterized using crystallographic approaches, which revealed that HbCLP2 was a novel isoform exhibiting an unusual half chitin-binding domain before the catalytic domain and displayed high thermostability and antifungal activity against Alternaria alternata (MartínezCaballero et al. 2014).

Table 2. Comparative analysis of chitinases across various sequenced plant genomes.

Serial No

1

2

3

Type

GH_18

GH_18 + Chit II

GH_19

Chlamydomonas

-

2

2

Physcomitrella

-

3

15

Selaginella

14

8

45

Oryz

31

2

19

Brachypodium

11

1

15

Zea

13

4

23

Sorghum

24

2

18

Vitis

13

9

24

Arabidopsis

1

9

14

Carica

10

1

6

Populus

15

11

28

Ricinus

8

4

8

Hevea

26

1

12

Glycine

27

7

15

Medicago

10

-

9

 

Conclusions

Our systematic analysis of the rubber genome sequence indicates that the number of chitinase genes in the genome, is similar to that found in other angiosperms. The present results provide the various classes of chitinases encoded in the rubber genome, for further study of their roles in defense, rubber biosynthesis and associated physiological activities in rubber tree. Although the spatio-temporal expression patterns, biochemical activities and molecular mechanisms regulated by chitinases need to be elucidated, this study provides important insights in the understanding of a novel and essential protein family with very little available functional information relevant to rubber tree. This study is a new starting point for further analysis of the physiological roles of the role of chitinases in plant defense responses, and sensu lato of disease resistance mechanisms.

Materials and methods

Genome-wide identification of chitinase encoding genes

The annotated protein sequences of H. brasiliensis genome (Rahman et al. 2013) were extracted from the GenBank assembly accession, GCA_000340545.1 (WGS: AJJZ010000001-AJJZ011223365, WGS_SCAFLD: KB611703-KB629728). Pooled sequences were screened against the Plant Genome Database for identification of possible ‘chitinase’ candidates’. InterPro domains specific to chitinases were searched in batch process [i.e., IPR001223 (Glyco_hydro_18 7982), IPR001579 (Glyco_hydro_18_chit_AS), IPR013781 (Glyco_hydro_subgr_catalytic) and IPR017853 (Glycoside_hydrolase_SF)] etc., using InterProScan (Quevillon et al. 2005) to retrieve the orthologs from rubber tree [Supplementary Table 5]. Post identification of 39 chitinases from rubber tree, IPR accessions were extracted by querying the BioMart [http://www.ebi.ac.uk/interpro/biomart/martview/]. Standalone version. BLASTP (Altschul et al. 1997) was carried out with NCBI database. Based on high bit score, lower E-value and maximum sequence identity (%), the best candidate hits were selected for further processing. The candidate sequences were curated manually using available annotations in SwissProt/TreEMBL, PDB, CDD, GenBank and existing literatures. Sequence comparisons with chitinases from other plants were performed using PSI-BLAST (Schaffer et al. 2010) at PRALINE webserver (Simossis and Heringa, 2005). GreenPhylDB v2.0 database (http://greenphyl.cirad.fr/v2/cgi-bin/index.cgi) was consulted for comparative genomics studies (Rouard et al. 2010) for plant chitinases.

Classification of the chitinase genes and analysis of conserved motifs

The identified genes were classified based on the presence of several Pfam domains, i.e., Conserved motif within the domains was identified using MEME (Multiple Expectation Maximization for Motif Elicitation) (Bailey and Elkan, 1995), while sequence logo were obtained after running GLAM2SCAN (Frith et al. 2008).

Mapping with Transcriptome and EST data

Annotated transcriptome sequences obtained from the H. brasiliensis strain RRIM 600 (Accession: PRJNA82895) and all available EST sequences (approx. 50,000) from the NCBI GenBank were extracted and pooled. The identified ‘putative’ chitinase sequences were mapped to transcriptome and EST sequences using BLASTp and hits accepted when greater than 95% sequence identity and the best alignment scores were. Based on the CDS features, the exon-intron structure of the entire gene family was visualized using the Gene Structure Display Server (GSDS) (Guo et al, 2007).

Sequence alignment and phylogenetic tree construction

Multiple alignments of amino acid sequences were done by Clustal X version 2.0 (Larkin et al. 2007). A phylogenetic tree was constructed using the Maximum Likelihood (ML) method (Saitou and Nei, 1987) with bootstrap multiple alignment resampling set at 1000 using Molecular Evolutionary Genetics Analysis (MEGA) software version 5.05 (Tamura et al. 2007). Multalin software (Corpet, 1988) was also used as a secondary method for aligning sequences with their secondary structures and rechecking results.

The WEGO tool was used to perform GO functional classification and plot all the 39 chitinases from H. brasiliensis genome assembly to view the distribution of gene functions of this family at the macro level. The analysis mapped all of the InterProScan annotated unigenes to GO terms in the database and computed the numbers associated with each term (Ye et al. 2006).

Calculation of physiochemical properties based on primary sequence

Amino acid composition the rubber tree chitinases were calculated using CLC protein workbench tool (www.clcbio.com/protein) and were expressed as percentage of hydrophobic and hydrophilic residues present in the protein. For physiochemical characterization, theoretical pI (isoelectric point), molecular weight, AI (aliphatic index) and GRAVY (grand average hydropathy) (Kyte and Doolittle, 1982) were computed using the Expasy's ProtParam server (Wilkins et al. 1999) for the set of proteins (http://us.expasy.org/tools/protparam.html). Prediction of allergenicity was done using the prediction web server at AllerHunter, a SVM pair-wise system for assessment of allergenicity (Muh et al. 2009). Additionally, allergenicity of the chitinases were also predicted at AlgPred web server (Saha and Raghava, 2006), as it follows predictions from SVMc, experimentally obtained IgE epitopes, allergen-representative peptides (ARPs) by BLAST and MEME/MAST motifs.

Competing interests

The author declares that there are no competing interests.

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