Table 2.
Complementation assays with AvrLm2 candidate gene. Phenotypic interaction of wild‐type and AvrLm2‐transgenic isolates on different B rassica lines harbouring Rlm2
| Isolates/transformantsb | B. napus lines/cultivarsa | ||||||
|---|---|---|---|---|---|---|---|
| Westar | Topas | T‐Rlm2 | Glacier | Bristol | Samourai | Tapidor | |
| Control | Control | Rlm2 | Rlm2,Rlm3 | Rlm2,Rlm9 | RLm2,Rlm9 | Rlm2 | |
| v23.1.3 (A1a2a3a9) | V | V | V | V | V | V | V |
| 00‐100 (a1A2A3A9) | V | V | A | A | A | A | A |
| v23.1.3: LmCys1(AW1) | V | V | A | A | A | A | A |
| v23.1.3: LmCys1(AW2) | V | V | A | A | A | A | A |
| v23.1.3: LmCys1(SNP1)c | V | V | V | V | V | V | V |
| v23.1.3: LmCys1(SNP2) | V | V | V | V | V | V | V |
| v23.1.3: LmCys1(SNP3) | V | V | V | V | V | V | V |
| v23.1.3: LmCys1(SNP4) | V | V | A | A | A | A | A |
| v23.1.3: LmCys1(Control) | V | V | A | A | A | A | A |
Pathogenicity test on differential lines/cultivar carrying different resistance genes. Each Leptosphaeria maculans isolate was tested on 12 seedlings of the differential lines and 12 seedlings of ‘Westar’ and Topas as highly susceptible controls. The disease reactions were scored 14 days after inoculation and rated using the 0–9 scale described by Williams (1985). T‐Rlm2 stands for Topas‐Rlm2.
Isolate v23.1.3 (A1a2a3a9) is avirulent on lines harbouring Rlm1, but virulent on lines harbouring Rlm2, Rlm3 and Rlm9, and isolate 00‐100 (a1A2A3A9) is virulent on the line harbouring Rlm1, but avirulent on lines harbouring Rlm2, Rlm3 and Rlm9. v23.1.3: LmCys1(AW1) is a transformant with the pNL11‐LmCys1 construct (LmCys1 avirulent allele was amplified from isolate 00‐100). V23.1.3: LmCys1(AW2) is a transformant with the pLM4‐LmCys1 construct (LmCys1 avirulent allele coding region was amplified from isolate 00‐100). All constructs were introduced into isolate v23.1.3 by Agrobacterium‐mediated transformation.
The four various alleles of AvrLm2 (single nucleotide polymorphisms, SNPs) were synthesized and cloned in a Gateway vector (pLM4) downstream of the promoter of the AvrLm1 effector. These SNPs are: SNP1, G397 → A397 (amino acid change Gly133 → Ser133); SNP2, G397 → C397 (amino acid change Gly133 → Arg133); SNP3, G398 → A398 (amino acid change Gly133 → Asp133); SNP4, G436 → C436 (amino acid change Glu146 → Gln146). The AvrLm2 open reading frame (ORF) from isolate 00‐100 was cloned in the same vector, as a positive control.