Figure 3.

Suppression of BAX‐induced cell death (CD) by Leptosphaeria maculans candidate effectors in Nicotiana benthamiana (Nb) leaves. Agrobacterium tumefaciens (Ag) strains expressing individual L. maculans small secreted proteins (SSPs) and Bax were infiltrated into Nb leaves. Ag carrying pGWB451‐GFP was used as a negative control. BAX‐induced cell death was scored at 4 days after infiltration. (A) Examples of complete suppression of CD by CE8 (jgi|Lepmu1|2216; Lema_T084480.1) and partial suppression of CD by CE5 (jgi|Lepmu1|4336; Lema_T100080.1), CE9 (jgi|Lepmu1|616; Lema_T006160.1) and CE14 (jgi|Lepmu1|9004; Lema_T038230.1) effectors. (B–D) Leptosphaeria maculans necrosis and ethylene‐inducing peptide‐1 (Nep‐1)‐like protein (NLP) (Lm‐NLP) are required for the complete virulence of Lm. (B) Expression of Lm‐NLP at different time points. (C) Transient expression of Lm‐NLP in Nb leaves induces hypersensitive response (HR)‐like cell death. Nb leaves were infiltrated with Ag cultures containing Lm‐NLP, Bax and green fluorescent protein (GFP). Bax and GFP served as positive and negative controls, respectively. Leaves were photographed 5 days after infiltration (left panel) and cell death was visualized by trypan blue staining of the same leaves (right panel). (D) Cotyledons of 7‐day‐old seedlings of Brassica napus cv. DH‐Topas 16516 were inoculated with the L. maculans wild‐type isolate 00‐100 and the three Lm‐NLP silenced (RNAi) lines (NLP‐SL1, NLP‐SL2 and NLP‐SL3), and were photographed at 14 days after inoculation.