Role of ACRE genes in induced resistance against Botrytis cinerea
In order to investigate whether other members of the ACRE gene family display a similar priming profile to ACRE75 , correlation analysis was performed on the subset of genes differentially expressed at 6 hpi. Genes with statistically significant similar profiles were identified (Table S3), which included ACRE180 at a confidence value of 0.956. In addition, analysis of the samples later in the experiment, confirmed that both ACRE75 and ACRE180 are primed also at later time points (Fig S4a,b).
In order to investigate whether primed expression of ACRE75 andACRE180 genes may be involved in enhanced disease resistance, genes from S. lycopersium and ortholog genes in N. benthamiana were overexpressed using both transient and stable systems. For SlACRE75 , best match against N. benthamiana genome was Niben101Scf03108g12002.1 (termed NbACRE75 ), sharing a 77.5% protein identity; (ii) For SlACRE180 , the best match against theN. benthamiana genome was Niben101Scf12017g01005.1 (termed NbACRE180), with 49.5% protein identity. Arabidopsis ortholog analysis failed to identify hits for ACRE75 and ACRE1280 candidate genes. Constructs were produced with a fused GFP protein in the N-terminus and protein integrity was confirmed via Western blot. Proteins extracted from N. benthamiana leaves 48h after agro-infiltration and Western blot analysis confirmed that they were the expected sizes (Fig S5). Subcellular location of proteins was analysed via confocal microscopy of GFP fluorescence. Overexpression constructs were co-infiltrated with RFP-marker pFlub vector (McLellan et al., 2013) (Fig S6a) into N. benthamiana reporter lines CB157 (nucleus mRFP marker - Fig S6b) and CB172 (ER mRFP marker - Fig S6c). Free GFP accumulated in both cytoplasm and nucleus (Fig S6d), whereas GFP-SlACRE75 and GFP-NbACRE75 fusions accumulated exclusively in the nucleus and nucleolus of N. benthamiana cells (Fig S6e,f). Furthermore, GFP-SlACRE180 fusion accumulated exclusively in ER (Fig S6g), whereas GFP-NbACRE180 fusion accumulation was exclusively in peroxisomes (Fig S6h).
To further investigate the impact of overexpression of ACRE genes in disease resistance, the 4 constructs containing GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE75 and GFP-NbACRE180, and GFP-empty vector (EV), were agro-infiltrated into leaves of N. benthamiana plants, which were subsequently challenged with B. cinerea . Chitosan-induced resistance against B. cinerea was proven effective in N. bethamiana (Fig 4a). All GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE75 and GFP-NbACRE180-infiltrated N. benthamiana leaves showed a significant decreased in B. cinereanecrotic lesion size compared with the EV control (Fig 4b). To further analyse ACRE75 and ACRE180 biological functions and to confirm their role in plant resistance against B. cinerea , Arabidopsis plants were transformed to constitutively overexpress GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE75 and GFP-NbACRE180 proteins. Homozygous lines were identified and growth phenotype of transgenic plants was analysed by measuring rosette perimeter. No statistically significant differences were identified (Fig S7). Five-week-old plants were infected with B. cinerea and disease was scored at 6 dpi. Transgenic GFP-SlACRE75, GFP-SlACRE180, GFP-NbACRE180 and GFP-NbACRE75 overexpression plants all showed an enhanced resistance phenotype and significantly decreased B. cinerea lesion sizes in comparison to Col-0 and GFP-EV controls (Fig 4c). Furthermore, GFP-SlACRE75 and its homolog GFP-NbACRE75-overexpression plants showed a stronger resistance to B. cinerea than GFP-SlACRE180 and GFP-NbACRE180 overexpression lines at 6 dpi (Fig 4c).