Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene-inducing peptides (NEPs)

Abstract

Successful host colonization by necrotrophic plant pathogens requires the induction of plant cell death to provide the nutrients needed for infection establishment and progression. We have cloned two genes encoding necrosis and ethylene-inducing peptides from Sclerotinia sclerotiorum, which we named SsNep1 and SsNep2. The peptides encoded by these genes induce necrosis when expressed transiently in tobacco leaves. SsNep1 is expressed at a very low level relative to SsNep2 during infection. The expression of SsNep2 was induced by contact with solid surfaces and occurred in both the necrotic zone and at the leading margin of the infection. SsNep2 expression was dependent on calcium and cyclic adenosine monophosphate signalling, as compounds affecting these pathways reduced or abolished SsNep2 expression coincident with a partial or total loss of virulence.

Figure 1

Sclerotinia sclerotiorum necrosis and ethylene‐inducing peptides (SsNEPs). (A) Alignment of SsNEP1 and SsNEP2 showing identical residues (white on black background), signal peptides (italics), conserved cysteine residues (!) indicative of Type I NEP1‐like proteins (NLPs) and the GHRHDWE motif (*). (B) Phylogenetic relationship between NLPs from various fungi and oomycetes. The proteins used in the analysis are coded with the first two letters representing the organism followed by the GenBank accession number. Af, Aspergillus fumigatus; As, Aspergillus nidulans; Ao, Aspergillus oryzae; Be, Botrytis elliptica; Fo, Fusarium oxysporum; Mg, Magnaporthe grisea; Pa, Pythium aphanidermatum NEP1; Pi, Phytophthora infestans; Pp, Phytophthora parasitica; Ps, Phytophthora sojae; Sn, Stagonospora nodorum; Ss, Sclerotinia sclerotiorum; Vd, Verticillium dahliae. Type I and Type II NLPs were classified according to Gijzen and Nürnberger (2006). Confidence values for each node are based on 100 bootstrap analyses.

Figure 2

Map of the Potato virus X‐based binary vector pgR107 used for the in planta expression of Sclerotinia sclerotiorum necrosis and ethylene‐inducing peptide genes (SsNep). Elements shown are: LB, left border; 35S, cauliflower mosaic virus 35S promoter; RdRp, viral RNA‐dependent RNA polymerase; M1–M3, viral movement proteins; CP, coat protein, Nos, Agrobacterium tumefaciens nopaline synthetase transcriptional terminator; RB, right border. Necrotic symptoms observed on Nicotiana benthamiana leaves 2 weeks after inoculation with PVX virus, PVX +SsNep1 or PVX +SsNep2.

Figure 3

Expression of Sclerotinia sclerotiorum necrosis and ethylene‐inducing peptide genes (SsNep) during infection of Brassica napus leaves. (A) Northern blot analysis showing expression in mycelia at various times (hours) after inoculation. The total RNA loaded in each lane is also shown. Lower panel shows SsNep1, SsNep2 and SsPg1 reverse transcriptase‐polymerase chain reaction (RT‐PCR) products amplified from mycelia 24 h after inoculation. Amplification of the corresponding loci using genomic DNA was conducted as a positive PCR control. (B) Division of the lesion into uninfected (U), margin (M) and necrotic (N) zones and Northern blot analysis showing expression in each zone.

Figure 4

Northern blot analysis showing the expression of Sclerotinia sclerotiorum necrosis and ethylene‐inducing peptide 2 gene (SsNep2) in mycelia in various liquid media or after transfer to solid surfaces in the presence or absence of glucose. The bottom panel shows the total RNA loaded in each lane.

Figure 5

The effect of compounds affecting cyclic adenosine monophosphate (cAMP) levels (caffeine) and calcium signalling on Sclerotinia sclerotiorum infection of Brassica napus leaves and expression of S. sclerotiorum necrosis and ethylene‐inducing peptide 2 gene (SsNep2). The top panels show leaves 16 h after inoculation (upper frame), as well as the lesions beneath (lower frame), for mycelia that were untreated (control) or treated with caffeine (2.5 and 20 mm), cyclosporin A (CspA; 4.9 and 9.9 nm), [ethylenebis(oxonitrilo)]tetraacetic acid (EGTA) (10 mm), lanthanum chloride (LaCl₃; 1 and 10 mm), compound 48/80 (5 µm) or compound U73122 (10 mm). The bottom panels show the effect of these compounds on new mycelial growth. A sample of the mycelial preparation used to inoculate the leaves was also placed in the centre of a minimal salts–glucose (MS‐Glu) plate and radial growth was assessed after 5 days.

Figure 6

Northern blot analysis showing the expression of Sclerotinia sclerotiorum necrosis and ethylene‐inducing peptide 2 (SsNep2) and SsActin genes in mycelia treated with compounds affecting cyclic adenosine monophosphate (cAMP) levels (caffeine) and calcium signalling and placed on B. napus leaves for 16 h. The bottom panel shows the total RNA loaded in each lane.

Figure 7

The effect of exogenous cyclic adenosine monophosphate (cAMP) application on Sclerotinia sclerotiorum infectivity and SsNep2 expression. (A) Lesions forming on Brassica napus leaves when mycelia from S. sclerotiorum strain 1980 and the sac1 adenylate cyclase mutant were treated with increasing levels of cAMP (mm) prior to inoculation. (B) Northern blot analysis showing SsNep2 expression 24 h after inoculation of B. napus leaves with mycelia treated with cAMP. The bottom panel shows the total RNA loaded in each lane.