Figure 1.
Co-production of MtNFP and MtLYK3 induces cell death in Nicotiana leaves.
A, The following MtNFP and MtLYK3 constructs were expressed alone or co-expressed in Nicotiana leaves: mock infiltration (1); MtNFP untagged+MtLYK3 untagged (2); MtNFP-sYFP2+MtLYK3-sYFP2 (3); MtLYK3-sYFP2 (4); MtLYK3 untagged (5); MtNFP-sYFP2 (6); MtNFP untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, MtDMI2-sYFP2 construct was expressed alone or co-expressed with either MtNFP-mCherry or MtLYK3-mCherry construct in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.
Figure 2.
Production of AtCERK1 in Nicotiana leaves induces cell death that requires AtCERK1 kinase activity.
AtCERK1-sYFP2 (A) and AtCERK1[K349E]-sYFP2 (B) constructs were expressed in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
Figure 3.
Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.
Agrobacterium transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD600 [MtNFP-3xFLAG] = 0.125 and OD600 [MtLYK3-3xFLAG] = 0.2 (A, B); OD600 [AtCERK1-3xFLAG] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
Figure 4.
MtNFP and MtLYK3, or AtCERK1 (co-)production in Nicotiana leaves induces defence-like responses.
A, Kinetics of cell death development in Nicotiana. Agrobacterium transformants carrying either MtNFP-3xFLAG or MtLYK3-3xFLAG construct were co-infiltrated into Nicotiana leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in Nicotiana leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of NbHIN1, NbPR1 basic, NbACRE31, and NbACRE132 expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]-3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of NbHIN1 (white columns), NbPR1 basic (grey columns), NbACRE31 (hatched columns), and NbACRE132 (black columns) normalized by one reference gene, MtEF1 α. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.
Figure 5.
Cell death upon MtNFP and MtLYK3 co-production in Nicotiana leaves does not require SmNF.
Agrobacterium transformants carrying either MtNFP-3xFLAG or MtLYK3-3xFLAG construct were co-infiltrated into Nicotiana leaves at a final concentration: OD600 [MtNFP] = 0.25 and OD600 [MtLYK3] = 0.4 (1); OD600 [MtNFP] = 0.15 and OD600 [MtLYK3] = 0.25 (2). Twelve hai parts of the transformed regions were syringe-infiltrated with 10−7 mM SmNF (circled in red) or DMSO diluted to the same concentration (circled in white). Macroscopic observation (left panel) and Evans blue staining (right panel) are depicted 33 hai. Bar is 1 cm.
Table 1.
Cell death induction upon (co-)expression of various RLK-encoding genes in Nicotiana leaves.
Table 2.
Cell death induction activity of MtNFP-sYFP2 truncated/mutated variants in Nicotiana leaves.
Table 3.
Cell death induction activity of MtLYK3-sYFP2 mutated variants in Nicotiana leaves.