Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis
- University of Tübingen, Germany
- University of Liege-Gembloux Agro-Bio Tech, Belgium
Figures
Figure 1
The Arabidopsis lysozyme 1 (LYS1) gene is transcriptionally activated upon pathogen-infection.
(A) LYM1 and LYM3 do not possess peptidoglycan (PGN) hydrolytic activity. Micrococcus luteus cell wall preparations were incubated with 20 μg affinity-purified His6-tagged LYM1 or LYM3 or 0.5 μg hen …
Figure 2
Analysis of LYS1 overexpression lines.
(A) RT-qPCR analyses of transcript levels in mature leaves of two independent transgenic lines expressing p35S::LYS1-GFP (LYS1OE-1, LYS1OE-2) relative to expression levels in wild-type. EF1α …
Figure 3 with 1 supplement
Analysis of LYS1 amiRNA lines.
(A) Predicted LYS1 gene structure (exons, black bars; introns, black lines; untranslated regions, gray). The region targeted by the amiRNA construct is indicated by an arrowhead. (B) Off-target …
Figure 3—figure supplement 1
Characterization of LYS1 T-DNA insertion lines.
(A) Predicted LYS1 gene structure (exons, black bars; introns, black lines; untranslated regions, gray). T-DNA insertion sites are indicated by triangles. (B) The T-DNA insertion lines (each two …
Figure 4 with 2 supplements
LYS1 is a glucan hydrolase.
(A-D) Protein extracts from adult wild-type or LYSOE-1 and LYSKD-1 homozygous lines were assayed for hydrolytic activity towards glycan substrates. Plants expressing secreted green fluorescent …
Figure 4—figure supplement 1
LYS1 is located in the plant apoplast.
(A) Apoplastic washes were prepared from leaves of wild-type Arabidopsis plants or the LYS1OE-1 and LYS1KD-1 lines. Apoplastic fluids (concentrated tenfold) or total leaf protein extracts were …
Figure 4—figure supplement 2
LYS1 is devoid of cellulose hydrolytic activity.
LYS1 was purified from 5-week-old LYS1OE plants and used for cellulase activity assays. The substrate 4-methylumbelliferyl-β-D-cellobioside was incubated for 1 hr with purified LYS1, commercial …
Figure 5
LYS1 transiently expressed in Nicotiana benthamiana possesses hydrolytic activity.
(A) Protein extracts from N. benthamiana leaves expressing LYS1 fused to the myc-epitope tag under control of the p35S promoter were separated on an SDS-polyacrylamide gel and analyzed by western …
Figure 6
Purified LYS1 generates immunogenic peptidoglycan (PGN) fragments.
LYS1 was purified from 5-week-old LYS1OE plants and used for PGN digestion. (A) 500 µg Bacillus subtilis PGN were digested for 7 hr with mutanolysin (50 µg/ml), native purified LYS1 (140 µg/ml), …
Figure 7
LYS1 lines are not impaired in resistance towards infection with Botrytis cinerea.
Five-week-old plants were infected with the necrotrophic fungus Botrytis cinerea. 5 μl spore suspension of 5 × 105 spores/ml was drop-inoculated on one half of the leaf; two leaves per plant were …
Figure 8
LYS1 mutation does not impinge on resistance towards Alternaria brassicicola.
Five-week-old plants were infected with the necrotrophic fungus Alternaria brassicicola. Six 5 μl droplets of a spore suspension of 5 × 105 spores/ml were inoculated on the leaf; two leaves per …
Figure 9 with 1 supplement
Manipulation of LYS1 levels causes hypersusceptibility towards bacterial infection and loss of peptidoglycan (PGN)-triggered immune responses.
(A and B) Transgenic LYS1 plants are hypersusceptible to bacterial infection. Growth of Pseudomonas syringae pv. tomato (Pto) DC3000 (A) or Pto DC3000 ΔAvrPto/AvrPtoB (B) was determined 2 or 4 days …
Figure 9—figure supplement 1
Impact of weak LYS1 overexpression.
(A) Transcript levels of LYS1 and the peptidoglycan (PGN) receptors LYM1, LYM3, and CERK1 in the strong LYS1 overexpressor line LYS1OE-1 compared with the weak overexpressor line LYS1OE-3. Total RNA …
Author response image 1
Author response image 1: Early defense responses are not enhanced in LYS1OE-lines.
(A) Ion leakage measured in leaves of WT, LYS1OE or LYS1KD lines at indicated time points after infiltration of Pto DC3000. (B) Determination of FRK1 transcript levels in seedlings of wild type …
Tables
Table 1
Primers used in this study
| AGI | Primer name | Sequence 5′ → 3′ |
|---|---|---|
| At5g24090 (LYS1) | At5g24090F1 | CCAGAGGTGGCATAGCCATC |
| At5g24090R1 | CATCTGGTGGGATATAGCCAC | |
| At5g24090F | ATGACCAACATGACTCTTCG | |
| At5g24090R | TCACACACTAGCCAATATAG | |
| At5g24090RP2 | TGATGCCACGAGACTGAC | |
| LP_N853931 | TGACGAACCATGATAAATGGG | |
| RP_N853931 | CATAACCTCACACTGTGCTCG | |
| LP_N595362 | TAGTGCATGCATGTTAAACCG | |
| RP_N595362 | AGCTCCTCAATGTCCATTTCC | |
| Salk-Lba | TGGTTCACGTAGTGGGCCATCG | |
| Ds5-1 | GAAACGGTCGGGAAACTAGCTCTAC | |
| Wisc-Lba (p745) | AACGTCCGCAATGTGTTATTAAGTTGTC | |
| At5g24090Fq | CACTTGCACCCATTTTGGC | |
| At5g24090Rq | CCTCGACCCAATCGAGTA | |
| At5g24090miR-s | GATTTGACGTAAGCATACCGCCCTCTCTCTTTTGTATTCC | |
| At5g24090miR-a | GAGGGCGGTATGCTTACGTCAAATCAAAGAGAATCAATGA | |
| At5g24090miR*s | GAGGACGGTATGCTTTCGTCAATTCACAGGTCGTGATATG | |
| At5g24090miR*s | GAATTGACGAAAGCATACCGTCCTCTACATATATATTCCT | |
| At5g24090gatF | AAAAAGCAGGCTACATGACCAACATGACTCTTCG | |
| At5g24090gatR | AGAAAGCTGGGTACACACTAGCCAATATAGATG | |
| At5g24090gatR-STOP | AGAAAGCTGGGTATCACACACTAGCCAATATAG | |
| At5g24090gatF2 | AAAAAGCAGGCTATGCCGTAGGCGAGTGTTTC | |
| At5g24090gatR2 | AGAAAGCTGGGTGTTTTTGGTTAAAGATGTTTG | |
| At1g07920/30/40(EF1α) | Ef1α-100-f | GAGGCAGACTGTTGCAGTCG |
| Ef1α-100-r | TCACTTCGCACCCTTCTTGA | |
| At2g19190 (FRK1) | FRK1-F | AAGAGTTTCGAGCAGAGGTTGAC |
| FRK1-R | CCAACAAGAGAAGTCAGGTTCGTG | |
| At4g02540 | At4g02540-qf1 | GTACCACGCCTATCTATT |
| At4g02540-qr1 | CTCATAGAAGAAACCAGCA | |
| At1g05615 | At1g05615-qf1 | GGATTCCTATCTCTACCT |
| At1g05615-qr1 | TTCTTTACCCTCATCAACC | |
| At5g58780 | At5g58780-qf1 | CTCTCTTCTCTTTTATCTCTCC |
| At5g58780-qr1 | CTCCTCCACTCCTACCACA | |
| At3g51010 | At3g51010-qf1 | GCGTCGTGCTTTTATACTG |
| At3g51010-qr1 | TTCTTCCTCTTCGCCTCT | |
| At1g21880 (LYM1) | Lym1-100-f | TACAACGGTATAGCCAACGGCACT |
| Lym1-100-r | GTGGAGCTAGAAGCGGCGCA | |
| At1g77630 (LYM3) | Lym3-100-f | ACTTCGCAGCAGAGTAGCTC |
| Lym3-100-r | AGCGGTGCTAATTGTTGCGG | |
| At3g21630 (CERK1) | CERK1-100-f | GGGCAAGGTGGTTTTGGGGCT |
| CERK1-100-r | CCGCCAAGAACTGTTTCGATGCC | |
| attB1 | GGGGACAACTTTGTACAAAAAAGCAGGCT | |
| attB2 | GGGGACCACTTTGTAC AAGAAAGCTGGGT |
