The circadian clock regulates receptor-mediated immune responses to an herbivore-associated molecular pattern

Abstract

Plants activate induced defenses through the recognition of molecular patterns. Like pathogen-associated molecular patterns (PAMPs), herbivore-associated molecular patterns (HAMPs) can be recognized by cell surface pattern recognition receptors leading to defensive transcriptional changes in host plants. Herbivore-induced defensive outputs are regulated by the circadian clock, but the underlying molecular mechanisms remain unknown. To investigate how the plant circadian clock regulates transcriptional reprogramming of a specific HAMP-induced pathway, we characterized the daytime and nighttime transcriptional response to caterpillar-derived In11 peptide, in the legume crop cowpea (Vigna unguiculata). Using diurnal and free-running conditions, we found that daytime In11 elicitation resulted in stronger late-induced gene expression than nighttime. Plants with a conditional arrhythmic phenotype in constant light (LL) conditions lost time-of-day dependent responses to In11 treatment, and this was associated with arrhythmic expression of circadian clock core transcription factor Late Elongated Hypocotyl VuLHY1 and VuLHY2. Reporter assays with VuLHY homologs indicated that they interact with the promoter of daytime In11-induced Kunitz Trypsin Inhibitor (VuKTI) via a canonical and a polymorphic CCA1/LHY Binding Site (CBS), consistent with a mechanism of direct regulation by circadian clock transcription factors. This study improves our understanding of the time-dependent mechanisms that regulate herbivore-induced gene expression.

Keywords: HAMP; In11; Inceptin; circadian clock; circadian gating; immunity; legumes; time-of-day.

Figure 1.. In11 induced responses are time-of-day dependent.

(a) Experimental design for RNA-seq. 14-day old cowpea plants grown under diurnal conditions (light/dark, LD) were treated (T) with wound + H₂O or wound + In11 at daytime (ZT4) or nighttime (ZT16), and samples were collected (C) 1h (ZT5 and ZT17) and 6h (ZT10 and ZT22) after treatment (n = 4 individual plants as biological replicates). (b) Volcano plots displaying the number of In11 down (↓) and upregulated (↑) genes (Log₂ Fold Change ∣LFC∣ ≥ 1 relative to wound + H₂O and p_adj < 0.05) 1h and 6h after treatment. (c) Venn diagram indicating the number of shared and unique differentially expressed genes (DEGs) 1h (ZT5 vs ZT17) and 6h (ZT10 vs ZT22) after daytime or nighttime treatment.

Figure 2.. Circadian clock related cis elements CBS and EE are present in the promoters of time-of-day dependent In11-induced defense genes.

(a) Scatter plot showing the Log₂ Fold Change (LFC) value of In11 DEGs (wound + In11 vs wound + H₂O) at 1h (ZT5 and ZT17) and 6h (ZT10 and ZT22) after treatment, and the absence/presence (gray/purple circles) of CBS or EE in their promoter (1.5 kb upstream start codon). The absolute value of the LFC difference (∣LFC_diff∣) is represented by the size of the circles, and selected defense-related genes are indicated. (b-c) Promoter structure and expression pattern of a Terpene Synthase (VuTPS) and Kunitz Trypsin Inhibitor (VuKTI) 6h (ZT10 and ZT22) after daytime and nighttime treatment. according to (b) RNAseq data (b) and qPCR data (c) are shown. Different letters indicate significant differences determined by two-way ANOVA followed by Tukey's Honest Significant Difference test (HSD) (n= 4-5 biological replicates, p-value < 0.05). Independent plants were sampled at each treatment - time combination.

Figure 3.. Cowpea Late Elongated Hypocotyl (VuLHY) homologs show typical cycling patterns and are downregulated by wounding.

(a) Maximum likelihood phylogenetic tree showing 14 LHY homologs from five legume species and Arabidopsis. Cowpea homologs VuLHY1 and VuLHY2 are highlighted in gray boxes. The scale bar indicates branch length as the mean number substitutions per site. Diurnal expression pattern of VuLHY1 and VuLHY2 according to (b) RNAseq (n = 4) and (c) qPCR data (n = 3-4 biological replicates). Samples were collected at ZT4, ZT5, ZT16, ZT17 and ZT22 from undamaged plants (gray), and 1(ZT5, ZT17) and 6 h (ZT10, ZT22) after daytime (ZT4) or nighttime (ZT16) wound + H₂O (orange) and wound + In11 (green) treatment. Independent plants were sampled at each treatment x time combination. Lines and error bars represent means ± SEM.

Figure 4.. Circadian expression patterns of VuLHY1, VuLHY2 and VuGI.

Expression patterns of (a) VuLHY1, VuLHY2 and (b) VuGI under constant light (LL) in cowpea trifoliates. Cowpea plants were grown under LD for 10 days and then transferred to LL. Leaf samples were taken every 4 hours over the course of four days for gene expression analyses. Lines and error bars represent means ± SEM (n = 3 biological replicates). Independent plants were sampled at each time point.

Figure 5.. Nighttime repression of In11-induced VuKTI is abolished in conditional arrhythmic cowpea plants.

(a) Experimental design. Cowpea plants were grown under light/dark (LD) for 10-13 days, and then transferred to constant light (LL) for one to four days. Plants were treated (T) by wound + H₂O (orange) or wound + In11 (green) 4 h after subjective dawn or subjective dusk, and samples were collected (C) 6 h later along with undamaged (gray) controls. (b) Expression pattern of VuKTI according to qPCR data. Different letters indicate significant differences determined by two-way ANOVA followed by Tukey's Honest Significant Difference test (HSD) (n= 3-6 biological replicates, p-value < 0.05) each day. Independent plants were sampled at each treatment - time combination.

Figure 6.. Cowpea LHY homologs modulate the activity of the VuKTI promoter in a CBS-dependent manner in tobacco.

Schematic representation of the firefly luciferase (LUC) reporters used in the N. benthamiana transient assay (a) Sequence of the CBS and CBS-like (CBS-L) cis-elements found in the KTI promoter. The CCA1/LHY binding site was mutated on CBS and CBS-L via site directed mutagenesis (underlined) (b) LUC reporters used in the assay. WT=CBS, CBS-L, M1 = ΔCBS, CBS-L, M2 = CBS, ΔCBS-L (c) The effect of the VuLHY1 and VuLHY2 proteins on the activity of the LUC reporters. At 72 h LUC activity was measured with 35S:LHY proteins co-expressed in a separate agrobacterium strain. Relative reporter activity was calculated by normalization against 35S:Renilla. Reporters final OD₆₀₀=0.3 and effectors final OD₆₀₀=0.4 Significant differences in the mean (*) were determined by a two-sided t-test of each effector vs. EV (n = 3-6 biological replicates, p-value < 0.05).