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. 2018 Oct 4;7:e38874. doi: 10.7554/eLife.38874

Figure 3. Chit5 encodes a root expressed class V chitinase with Nod factor hydrolase activity.

(A) Chit5 gene structure. Mutation in chit5-2 allele is shown. Chit5 gene is deleted in chit5-1 and chit5-3 alleles. (B) Chit5 promoter activity was monitored in Gifu roots transformed with a Chit5 promoter-tYFP-NLS (green nuclei) in uninoculated and M. loti R7A + DsRed inoculated roots. White arrows highlight examples of uninfected cells showing Chit5 promoter activity. Scale bars are 200 µm. (C) ROS induced by exudates of CO-VI-treated plants. (D) Absence of ROS induction by exudates of M. loti R7A Nod factor-treated plant exudates. (E) M. loti R7A Nod factor hydrolysis in the presence of the indicated plant genotypes measured by HPLC-MS. The average relative percentage of LCO-V and LCO-II fractions from two biological replicates (Figure 3—source data 1) determined from peak-peak integration is indicated in brackets. The peak eluting near 4.1 min is present in all plant treated samples but was not detected in the control sample. It lacks the characteristic fragmentation pattern of LCOs, and appears to be an aromatic, small molecule unrelated to chitinase activity.

Figure 3—source data 1. HPLC-MS analysis of Nod factor isolated after exposure to roots of wild-type Gifu or chit5 mutants.
elife-38874-fig3-data1.docx (13KB, docx)
DOI: 10.7554/eLife.38874.017

Figure 3.

Figure 3—figure supplement 3. Complementation of chit5 mutant alleles with Chit5.

Figure 3—figure supplement 3.

Gifu (A) and chit5 mutant plants (B–D) were transformed with an empty vector (EV) control or Chit5 expressed from its native or Ljubiquitin promoter. Plants were harvested 6 weeks post-inoculation with M.loti R7A. Similar results were obtained using the two promoters and images show three representative plants and examples of the nodules formed. Scale bars are 1 cm for plant images and 1 mm for nodules.

Figure 3—figure supplement 4. Chit5 promoter-GUS reporter analysis .

Figure 3—figure supplement 4.

Gifu plants transformed with a Chit5 promoter-GUS reporter construct were analysed after inoculation with M. loti R7A + DsRed. (A) Chit5 promoter activity in transformed roots uninoculated or inoculated with M. loti R7A + DsRed at the indicated time points. Scale bars are 0.5 mm. (B) Upper panel shows light microscopy of GUS-reporter staining in nodules at different developmental stages and the lower panel shows M. loti R7A + DsRed colonization state of these same nodules Note the increase in DsRED fluorescence in the nodules with reduced GUS activity. Scale bars are 0.5 mm. (C) Sections of mature nodules show that Chit5 promoter activity is restricted to non-colonized nodule cells. Scale bars are 200 μm.

Figure 3—figure supplement 5. Amino acid alignment of CHIT5, AtCHIC, MtNFH1.

Figure 3—figure supplement 5.

Amino acid sequence alignment of Lotus japonicus CHIT5, Medicago truncatula NFH1 (KC833515) and Arabidopsis thaliana CHIC (NP_193716). (*) indicates a conserved residue, (:) indicates strongly similar properties, (.) indicates weakly similar properties, (-) indicates gaps. A predicted signal peptide is indicated in bold for CHIT5. The conserved catalytic motif DxDxE is indicated as is the position of the E-K mutation (green) introduced to produce an inactive CHIT5 construct. The position of the P-L substitution in chit5-2 is indicated (magenta). The position of A and B loops (in red) and of the proline residue (red box) identified as important for Nod factor hydrolase activity of MtNFH1 are indicated.

Figure 3—figure supplement 6. Complementation of chit5 mutant alleles with Chit5 (E166–K).

Figure 3—figure supplement 6.

Gifu (A) and chit5 mutant plants (B–D) were transformed with an empty vector (EV) control or an active site mutant version of Chit5 (E to K) expressed from its native promoter. Plants were harvested 6 weeks post-inoculation with M. loti R7A. Images show three representative plants for each transformation construct and examples of the nodules formed. Scale bars are 1 cm for plant images and 2 mm for nodules.

Figure 3—figure supplement 7. Several Lotus japonicus chitinase genes are expressed in roots.

Figure 3—figure supplement 7.

(A) Phylogenetic analysis of protein sequences of annotated chitinases or glycosyl hydrolases from Lotus genome v3.0. (B) Log10 total counts from RNA-seq analysis performed on Gifu roots and shoots 3 dpi with water or M.loti R7A from Lotus base (www.lotus.au.dk). Chit5 (as assigned in v3.0) is highlighted in red.

Figure 3—figure supplement 2. Alignment of Chit5 paralogs.

Figure 3—figure supplement 2.

Nucleotide sequence alignment of Chit5 and its two paralogs (start to stop codons). Exons are highlighted in green and nucleotide polymorphisms are shown in red. The premature stop codons in the paralogs are highlighted by red boxes and the position of the C to T point mutation in chit5-2 is indicated with an asterisk.

Figure 3—figure supplement 1. Map-based cloning of Chit5 gene.

Figure 3—figure supplement 1.

(A) Positions of bacterial artificial chromosome (BAC) and transformation-competent artificial chromosome (TAC) clones from L. japonicus MG20 and BAC clone 69G19 from L. japonicus Gifu are shown. The closest markers with the number of informative recombinations delimiting the Chit5 locus are indicated. A schematic representation of the three Chit5 paralogs within the delimited region is shown with PCR primer binding sites indicated. (B) Amplicon detection in the indicated genotypes using primers specific for Chit5 (P1L/P1R) and common for the three Chit5 paralogs (P2L/P2R).