Eucalyptus grandis (W. Hill ex Maiden) is an Australian Myrtaceae tree grown for timber in many parts of the world and for which the annotated genome sequence is available. Known to be susceptible to a number of pests and diseases, E. grandis is a useful study organism for investigating defense responses in woody plants. Chitinases are widespread in plants and cleave glycosidic bonds of chitin, the major structural component of fungal cell walls and arthropod exoskeletons. They are encoded by an important class of genes known to be up-regulated in plants in response to pathogens. The current study identified 67 chitinase gene models from two families known as glycosyl hydrolase 18 and 19 (36 GH18 and 31 GH19) within the E. grandis genome assembly (v1.1), indicating a recent gene expansion. Sequences were aligned and analyzed as conforming to currently recognized plant chitinase classes (I–V). Unlike other woody species investigated to date, E. grandis has a single gene encoding a putative vacuolar targeted Class I chitinase. In response to Leptocybe invasa (Fisher & La Salle) (the eucalypt gall wasp) and Chrysoporthe austroafricana (Gryzenhout & M.J. Wingf. 2004) (causal agent of fungal stem canker), this Class IA chitinase is strongly up-regulated in both resistant and susceptible plants. Resistant plants, however, indicate greater constitutive expression and increased up-regulation than susceptible plants following fungal challenge. Up-regulation within fungal resistant clones was further confirmed with protein data. Clusters of putative chitinase genes, particularly on chromosomes 3 and 8, are significantly up-regulated in response to fungal challenge, while a cluster on chromosome 1 is significantly down-regulated in response to gall wasp. The results of this study show that the E. grandis genome has an expanded group of chitinase genes, compared with other plants. Despite this expansion, only a single Class I chitinase is present and this gene is highly up-regulated within diverse biotic stress conditions. Our research provides insight into a major class of defense genes within E. grandis and indicates the importance of the Class I chitinase.
S1 Fig. Domains and classes for glycoside hydrolase 18 and 19 (chitinases) from the Eucalyptus grandis sequences. S = signal sequence, H = hinge region (proline/threonine-rich in Class IV and glycine-rich in Class I), CBD = chitin binding domain, black box indicates C-terminal vacuolar extension, aa = amino acid residue with approximate sizes. Diagrammatic concept from Collinge, Kragh et al. (1993).
S2 Fig. Quantitative reverse-transcriptase polymerase chain reaction relative expression change (A) of putative Class 1A chitinase (Eucgr. I01495) in Eucalyptus grandis in response to Chrysoporthe austroafricana inoculation (three days post-inoculation). Expression is relative to reference gene Elongation factor S-II (B). R = resistant, S = susceptible.
S3 Fig. Predicted tertiary structure for (A) Eucalyptus grandis Class IA chitinase peptide with cleaved signal and vacuolar sequences had a C-score of 1.72 (with range being between 5 and 2) and estimated TM-score of 0.96 (structural similarity score between 0 and 1) (Roy et al. 2010). (B) The putative mature protein matched tertiary structure for Oryza sativa L. japonica Class IA chitinase crystal structure (Kezuka et al. 2010) with TM score of 0.95 (http://www.rcsb.org/pdb/explore/jmol.do?structureId=2DKV&bionumber=1&opt=3&jmolMod e=HTML5). Blue = GH19 and Chitin binding domains (linked by hinge region), white = hinge region (glycine-rich in E.grandis Class I but usually proline/threonine rich), red = catalytic regions as identified by 0.6nm of bound substrate, magenta = essential residues for catalytic activity determined with mutagenesis (Bishop et al. 2000).
S1 Table. Full list of Eucalyptus grandis putative chitinase genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and localization. Log2 of normalized FPKM reads expression data, log2 fold change values and ANOVA results (p values). S_F_C = susceptible, fungal treatment, control. S_F_I = susceptible, fungal treatment, inoculated. R_F_C = resistant, fungal treatment, control. R_F_I = resistant, fungal treatment, inoculated. S_I_C = susceptible, insect treatment, control. S_I_I = susceptible, insect treatment, infested. R_I_C = resistant, insect treatment, control. R_I_I = resistant, insect treatment, infested.