Abstract
As an endoplasmic reticulum (ER) stress sensor, inositol-requiring enzyme 1 (IRE1) splices the bZIP60 mRNA, and produces an active bZIP60 transcription factor that regulates genes involved in the unfolded protein response (UPR) during ER stresses. This IRE1-bZIP60 pathway is conserved in plant species and recently implicated in plant-pathogen interaction. However, it is unclear whether this IRE1-bZIP60 pathway is involved in Nicotiana attenuata resistance to necrotic fungal pathogen, Alternaria alternata. In this study, transcriptional levels of chaperone protein genes, including luminal binding protein (BiP), protein disulfide isomerase (PDI), calnexin 1-like (CNX 1-like), and calreticulin (CRT), and genes involved in IRE1-bZIP60 pathway, were all significantly induced in N. attenuata leaves after A. alternata inoculation. Silencing IRE1 or bZIP60 led to N. attenuata plants more susceptible to A. alternata, which were associated with reduced gene expressions of Feruloyl-CoA 6′-hydroxylase 1 (F6′H1), a gene encoding a key enzyme for phytoalexin scopoletin and scopolin biosynthesis. Further, electromobility shift assays (EMSA) indicated that bZIP60 protein of spliced form could directly bind to the promoter region of F6′H1 in vitro. JA signaling pathway is required for N. attenuata resistance to A. alternata. Interestingly, the fungus-elicited transcriptional levels of BiP, PDI, CNX 1-like, CRT, IRE1, and bZIP60(s) were all significantly decreased in JA-deficient or JA-insensitive plants. Meanwhile, those genes were significantly induced by methyl jasmonate (MeJA) when applied exogenously. However, the transcriptional levels of JA-regulated genes allene oxide synthase (AOS) and lipoxygenease 3 (LOX3) were not affected in plants impaired with IRE1-bZIP60 pathway. Thus, it is concluded that IRE1-bZIP60 pathway is required for N. attenuata resistance to A. alternata, and JA signaling pathway plays an important role in the elicitation of chaperone protein genes and IRE1-bZIP60 pathway.
Highlights
The endoplasmic reticulum (ER) is a major protein-folding organelle in cells (Ellgaard and Helenius, 2003; Trombetta and Parodi, 2003)
Mean (±SE) binding protein (BiP) (A), protein disulfide isomerase (PDI) (B), CNX 1-like (C), and CRT (D) transcripts were measured by real-time PCR in five biological replicates of source-sink transition leaves treated with mock infection of A. alternata at 1 dpi
Mean (±SE) IRE1a (A), IRE1b (B), and bZIP60(s) (C) transcripts were measured by real-time PCR in five biological replicates of source-sink transition leaves treated with mock infection of A. alternata at 1 dpi
Summary
The endoplasmic reticulum (ER) is a major protein-folding organelle in cells (Ellgaard and Helenius, 2003; Trombetta and Parodi, 2003). The IRE1/bZIP60 pathway suppressed systemic accumulation of potyviruses and potexviruses in Arabidopsis and N. benthamiana (Gaguancela et al, 2016) It is unclear whether UPR is involved in plant resistance to necrotrophic fungal pathogens. The necrotrophic fungal pathogen Alternata alternata (tobacco pathotype) causes brown spot disease, which is one of the most common and destructive fungal diseases of Nicotiana species In response to this fungal infection, phytoalexins scopoletin and scopolin and transcripts of their key enzyme gene feruloyl-CoA 6′-hydroxylase 1 (F6′H1) are highly elicited in N. attenuata leaves around the attacked sites (Sun et al, 2014; Li and Wu, 2016). The role of IRE1bZIP60 pathway in plant resistance was tested in plants silenced with either IRE1 or bZIP60 via virus-induced gene silencing (VIGS), and the relation among IRE1-bZIP60 pathway, F6′H1, and JA signaling pathway was explored in this study
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