IQD1 Involvement in Hormonal Signaling and General Defense Responses Against Botrytis cinerea

Abstract

IQ Domain 1 (IQD1) is a novel Arabidopsis thaliana calmodulin-binding protein, which was found to be a positive regulator of glucosinolate (GS) accumulation and plant defense responses against insects. We demonstrate here that the IQD1 overexpressing line (IQD1 ^OXP ) was also more resistant also to the necrotrophic fungus Botrytis cinerea, whereas an IQD1 knockout line (iqd1-1) was much more sensitive. Furthermore, we showed that IQD1 is up-regulated by jasmonic acid (JA) and downregulated by salicylic acid (SA). A comparison of whole transcriptome expression between iqd1-1 and wild type plants revealed a substantial downregulation of genes involved in plant defense and hormone regulation. Further examination revealed a marked reduction of SA and increases in the levels of ethylene, JA and abscisic acid response genes in the iqd1-1 line. Moreover, quantification of SA, JA, and abscisic acids in IQD1 ^OXP and iqd1-1 lines relative to the wild type, showed a significant reduction in endogenous JA levels in the knockout line, simultaneously with increased SA levels. Relations between IQD1 ^OXP and mutants defective in plant-hormone response indicated that IQD1 cannot rescue the absence of NPR1 or impaired SA accumulation in the NahG line. IQD1 cannot rescue ein2 or eto1 mutations connected to the ethylene pathway involved in both defense responses against B. cinerea and in regulating GS accumulation. Furthermore, IQD1cannot rescue the aos, coi1 or jar1mutations, all involved in the defense response against B. cinerea and it depends on JAR1 to control indole glucosinolate accumulation. We also found that in the B. cinerea, which infected the iqd1-1 mutant, the most abundant upregulated group of proteins is involved in the degradation of complex carbohydrates, as correlated with the sensitivity of this mutant. In summary, our results suggest that IQD1 is an important A. thaliana defensive protein against B. cinerea that is integrated into several important pathways, such as those involved in plant defense and hormone responses.

Keywords: Botrytis cinerea; IQD1; defense responses; glucosinolates; hormone signaling.

FIGURE 1

Pathogenicity of B. cinerea to Arabidopsis plants. Shown are averages of lesion size (mm²) of B. cinerea (Grape) on parental WT plants (Ws-0 or Col-0), on an IQD1 knockout line iqd1-1 and the over expressor line IQD1^OXP 72 h post-inoculation. Each column represents an average of 20 leaves, with standard error bars indicated. Asterisks above the columns indicate statistically significant differences at P < 0.05 from the corresponding WT, as determined using Student’s t-test. Results shown are from a biological replicate representative of six independent experiments.

FIGURE 2

Differentially expressed clusters and genes in iqd1-1 vs. WT plants. Enriched annotation terms of functional-related genes were grouped into clusters using the DAVID bioinformatics resources website. Positive enrichment scores denote upregulated clusters in iqd1-1 lines, while negative values denote up-regulated clusters in WT plants. (A) Differentially expressed clusters and genes in iqd1-1 vs. WT plants. (B) Differentially expressed clusters in infected iqd1-1 vs. infected WT plants. (C,D). A MapMan regulation overview map showing differences in transcript levels between iqd1-1 and WT plants. Red squares represent higher gene expression in mock treated iqd1-1 plants while blue squares represent higher gene expression in mock-treated WT plants, A regulatory network is presented in (C), while stress response network is shown in (D).

FIGURE 3

Elicitors affect IQD1 expression. Transgenic seedlings of gene trap line IQD1pro:GUS were treated with 100 μM salicylic acid, 100 nM Flg22, 100μM jasmonic acid, 500 μg/ml chitin or an equal volume of water as control for 18 h prior to histochemical GUS staining (A) or RNA extraction followed by qRT-PCR (B). Results shown are from a biological replicate representative of six independent experiments for GUS staining and three for qRT-PCR. (C) SA, JA, and ABA accumulation in IQD1 mutants. Plant hormones were extracted from 3 week-old Arabidopsis seedlings grown on half-strength MS agar plates. Quantitative analysis of plant hormones was accomplished using LC-MS/MS with isotopically labeled analogs serving as internal standards. Each column represents an average of three independent biological replicates, with standard error bars indicated. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using Tukey’s honest significant difference test. Asterisks above the columns indicate statistically significant differences relative to WT plants at P < 0.05, as determined using Student’s t-test.

FIGURE 4

IQD1^OXP affects SA pathway mutants. (A) Detached leaves from 6 week-old Arabidopsis SA pathway mutants were inoculated with B. cinerea. Lesion sizes were measured 72 h post-inoculation. Average lesion sizes from 30 leaves of each line are presented, along with the standard error of each average. All numbers are presented as the relative percentage to the corresponding background wild-type. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using the Tukey’s honest significant difference test. (B) Glucosinolates were extracted from 6-week old Arabidopsis seedlings of SA pathway mutants and analyzed by HPLC. Mean contents of methionine-derived (black bars) and tryptophan-derived (gray bars) glucosinolates are given for each line. Each column represents an average of eight seedlings, with standard error bars indicated. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using Tukey’s honest significant difference test. Results shown are from a biological replicate representative of three independent experiments.

FIGURE 5

IQD1^OXP affects JA pathway mutants. (A) Detached leaves from 6 week-old Arabidopsis JA pathway mutants were inoculated with B. cinerea. Lesion sizes were measured 72 h post-inoculation. Average lesion sizes from 30 leaves of each line are presented along with the standard error of each average. All numbers are presented as the relative percentage to their corresponding background wild-type. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using Tukey’s honest significant difference test. (B) Glucosinolates were extracted from seedlings of 6 week-old Arabidopsis SA pathway mutants and analyzed by HPLC. Mean contents of methionine-derived (black bars) and tryptophan-derived (gray bars) glucosinolates are given for each line. Each column represents an average of eight seedlings, with standard error bars indicated. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using Tukey’s honest significant difference test. Results shown are from a biological replicate, representative of three independent experiments.

FIGURE 6

IQD1^OXP effect on ethylene pathway mutants. (A) Detached leaves from 6 week-old Arabidopsis ethylene pathway mutants were inoculated with B. cinerea. Lesion sizes were measured 72 h post-inoculation. Average lesion sizes from 30 leaves of each line are presented along with the standard error of each average. All numbers are presented as the relative percentage to their corresponding background wild-type. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using Tukey’s honest significant difference test. (B) Glucosinolates were extracted from 6-week old Arabidopsis seedlings of SA pathway mutants and analyzed by HPLC. Mean contents of methionine-derived (black bars) and tryptophan-derived (gray bars) glucosinolates are given for each line. Each column represents an average of eight seedlings with standard error bars indicated. Different letters above the columns indicate statistically significant differences at P < 0.05, as determined using Tukey’s honest significant difference test. Results shown are from a biological replicate, representative of three independent experiments.

FIGURE 7

GO enrichment analysis of up-regulated B. cinerea genes. Significantly enriched GO terms classified by biological process, molecular function and cellular component when infecting iqd1-1 (A) or WT plants (B). Only GO terms that applied to more than 20 differentially expressed genes are shown.

FIGURE 8

Classification of CAZymes encoded by up-regulated DEGs. The contribution of each CAZymes family is shown. Numbers in brackets denote the number of DEGs for each family.

FIGURE 9

Suggested model of IQD1 involvement in glucosinolate accumulation and defense responses. Intact arrows indicate positive connections (→), intact lines with diamond heads indicate negative connections (→), and dashed lines indicate effects on expression (…..). JA-Il, jasmonic acid isoleucine; aGS, aliphatic glucosinolate; iGS, indolic glucosinolate; JAR1, JASMONATE RESISTANT 1; NPR1, NON-EXPRESSER OF PR GENES1; EIN2, ETHYLENE INSENSITVE 2.