Os04g0494100

GeneOs04g0494100 ,namelyOsChia4a,is a class IV chitinase that has similarity (about 70z identity) to both maize ChiA and Arabidopsis ChIV of class IV chitinases.

Annotated Information

Function

Fig. 1 Mutation analysis of candidate cis regulatory elements in the promoter. (A) Nucleotide sequence of the region from −515 to −265 upstream of the ATG translation initiation site of OsChia4a. The three E-boxes are denoted by E1–E3. (B) Mutated E-boxes (indicated by X), in which the CANNTG sequence was altered to AANNTG, were used for the dual LUC assays as performed in Fig. 3. In each construct (m1, m2 and m3), mutation was introduced into each E-box (E1, E2 and E3), respectively. Black and open bars indicate the relative LUC activities (FLUC/RLUC) of the deletion derivatives after 12 h of incubation of the rice cells with and without 100 μM JA, respectively. The data are expressed as mean values plus/minus the standard deviation of three replicates. A statistical analysis was performed using the t-test, and the significant differences (p < 0.01) between the relative LUC activities in the treated and in the untreated cells were denoted by asterisks.

Fig. 2 Family 19 Chitinases of Nipponbare and Other Cultivars .

Fig. 3Antifungal activity of purified OsChia4a-His protein. (A) Purified OsChia4a-His protein (lane P) was analysed by 12% SDS-PAGE along with molecular standards (lane M); it is shown by Coomassie Brilliant Blue staining. Arrowhead shows the purified OsChia4a-His protein (31.4 kDa). (B) A typical image of hyphae elongation of washed conidia of M. oryzae, which were mixed with purified OsChia4a-His protein or bovine serum albumin (BSA). After incubation for one day at 28 °C, the growth of M. oryzae was examined under the microscope. (C) Hyphal length of incubated conidia as shown in (B) was measured. The results are the average of more than 100 conidia examined in each condition; bars indicate the standard error from the mean value. .

• OsChia4a, which was identified to be one of the highest JA-inductive genes. The recombinant protein of His-tagged OsChia4a exhibited an inhibitory effect against the spore germination and hyphal growth of Magnaporthe oryzae .The promoter analysis of OsChia4a revealed that the region from −515 bp to −265 bp upstream of the ATG translation initiation site was required for the responsiveness to JA. A subsequent mutation analysis indicated that an E-box (CANNTG) in this region act as a JA-responsive cis element. These results imply that a basic helix-loop-helix transcription factor is likely to be involved in the regulation of the OsChia4a expression in a JA-dependent manner(Fig. 1).
  
  
• The high sequence conservation (95z identity) between the catalytic domains of OsChia4a (class IV) and OsChia2b (class II) provides an exam- ple of the direct and recent diversion of their genes via either deletion or insertion mechanisms within rice.The class IV catalytic domains have three deletions that correspond to the loops between a- helices C and D, F and G, and G and H.These loops do not appear to signicantly contribute to stability and activity towards chitin polymers (Fig. 2).
  
  
• OsChia4a is one of the highest induced chitinase genes (in terms of fold induction compared to the reference, untreated sample) both in the leaves and in the suspension cell. Unlike class IV, class I chitinases, OsChia1a and OsChia1c, which have been reported to be induced by the chitin elicitor in suspension cells, were only induced in the leaves and not in the suspension cells after the JA treatment .
  
  
• The antifungal activity of OsChia4a was demonstrated using the recombinant His-tagged OsChia4a protein that was produced in N. benthamiana using the Cowpea mosaic virus vector system .The protein was purified by using MagneHis Protein Purification System (Promega) ( Fig. 1A) and subjected to the hyphal extension-inhibition assay against M. oryzae as described in section “Materials and methods”(Fig. 3).
  
  

Expression

Fig. 4 Expression analysis of OsChia4a in suspension-cultured rice cells treated with 100 μM JA. The mRNA levels were analysed by SYBR green-based real-time RT-PCR. The mRNA level of OsChia4a was normalised to the levels of the Ubiquitin. The result was shown as a relative amount to untreated control (0 h). The results are the average of at least three independent experiments; bars indicate the standard error from the mean value. .

• The fragment of OsChia4a ORF without the stop codon was inserted into pEAQ-HT using the Nru I and Sma I sites to generate the C-terminal His-tagged protein. The infiltration of N. benthamiana with Agrobacterium was performed as previously described . The protein was extracted from the N. benthamiana leaves and OsChia4a-His protein was purified using MagneHis Protein Purification System , according to the manufacturer's instructions .E-box is recognised by a bHLH transcrip- tion factor, OsChia4a gene expression might be regulated by RERJ1.
  
  
• The raw data from qRT-PCR were analysed using the ΔCT (the difference in the threshold cycles) method, and the mRNA level of OsChia4a was normalised to the levels of the Ubiquitin. The result was shown as a relative amount to untreated control (0 h). The following oligonucleotide sequences were used: Ubiquitin, 5′-TCCGAGAGATGGGTTTCATC-3′, and 5′-GCCAAGATTGCCAAGAAGAC-3′ as well as OsChia4a, 5′-CGGCTCATCGATCAGGACT-3′, and 5′-CATTTAATCCTGATCCCAACATT-3′.
  
  
• A time course analysis of the OsChia4a expression was performed by qRT-PCR in suspension-cultured rice cells treated with JA. OsChia4a mRNA was already detected in the untreated sample (0 h) , suggesting that this chitinase is expressed to some extent in the non-stressed condition. After the JA treatment, the OsChia4a mRNA started to increase within 2 h from the treatment and reached a maximum level at 6 h after the treatment. The expression was maintained at a high level for 24 h after the treatment, with only a slight decrease "(Fig. 4).
  
  

Evolution

Fig. 5 Schematic Structures (A) and Structural Relationship (B) of Rice Family 19 Chitinases. .

• Belongs to family 19 chitinases in Oryza sativa L. cv. Nipponbare: one class I (OsChia1d), two class II (OsChia2a and OsChia2b), and one class IV (OsChia4a). OsChia2a resembled (about 60z identity) the catalytic domains of class I chitinases, but OsChia2b was almost identical (95z identity) to that of the class IV enzyme(Fig. 5).
  
  
• Sequencing the cDNA inserts (Æ1 kb) with similarity to known class I chitinase in the EST library (48,526 clones) of the Rice Genome Project (http: /rgp.aŠrc.go.jp) discovered four new family 19 chitinases named OsChia1d (DDJB accession num- ber AB096139), OsChia2a (AB016497), OsChia2b (AB003194), and OsChia4a (AB096140).OsChia2b and the catalytic domain of OsChia4a were very similar (95z identity) (Fig. 1B) and resembled (60–70z identity) the class IV chitinases of other plants rather than the class I enzymes (o60 identity) .
  
  
• OsChia4a, is a class IV chitinase that has similarity (about 70z identity) to both maize ChiA and Arabidopsis ChIV of class IV chitinases.34,35) This notion is in accordance with the hypothesis that the class I and IV genes diverged before the separation of dicots and monocots.
  
  

Labs working on this gene

• Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
• National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
• Department of Biosystem Science, Graduate School of Science and Technology, Niigata University, 8050 Ikarashi-2, Niigata 950-2102, Japan
• John Innes Centre, Norwich NR4 7UH, United Kingdom
• Département de Phytologie, Université Laval, Québec G1V 0A6, Canada