IQD1 is involved in glucosinolate accumulation and in general defense responses. JA activates IQD1 requires functional JA/ET and SA signaling pathways to control glucosinolate accumulation and defend against Botrytis cinerea and depends on JAR1 to control indole glucosinolate accumulation.

Plants must continuously adapt and protect themselves both against abiotic stressors, such as drought, extreme temperatures, improper lighting and excessive salinity and biotic stress imposed by other organisms such as viruses, bacteria, fungi and insects. Plants are resistant to most pathogens and in spite their sessile nature they have evolved a wide variety of constitutive and inducible defense mechanisms. Constitutive defenses include pre-formed physical barriers composing of cell walls, a waxy epidermal cuticle, bark and resins (Heath, 2000a). If this first line of defense is breached, then the plant must resort to a different set of chemical mechanisms in the form of toxic secondary metabolites and antimicrobial peptides, which are ready to be released upon cell damage (Tam et al., 2015). These pre-formed compounds are either stored in their biologically active forms like saponins (Podolak et al., 2010), or as precursors that are converted into toxic anti-microbial molecules only after pathogen attack, as exemplified by the glucosinolate-myrosinase system (Wittstock and Halkier, 2002). Other defense responses require detection of the invading pathogen by the plant and activation of inducible responses, often culminating in deliberate localized cell suicide in the form of the hypersensitive response (HR) so as to limit pathogen spread (Gilchrist, 1998; Heath, 2000b). Plants activate local defenses against invading pathogens within minutes or hours, with levels of resistance in distal tissue being influenced by systemic signals mediated by plant hormones. The identity of the pathogen determines the type of systemic response. The classic dogma holds that jasmonic acid (JA) and ethylene signaling activate resistance against necrotrophs whereas the salicylic acid (SA) signaling pathway is important for fighting biotrophic pathogens, although it also plays some role in defending against the necrotrophic fungi Botrytis cinerea (Govrin and Levine, 2000; Ferrari et al., 2003; Vuorinen et al., 2021). These two pathways are mostly antagonistic, with the balance of crosstalk between them affecting the outcome of the pathology (Glazebrook, 2005). Botrytis cinerea causes disease in more than 200 plant species including numerous economically important crops such as tomatoes and grapes (AbuQamar et al., 2016). The fungus has a predominantly necrotrophic lifestyle that involves killing plant host cells by diverse phytotoxic compounds and degrading enzymes, after which it extracts nutrients from the dead cells. It comprises nearly 300 genes encoding proteins considered Carbohydrate-Active enZymes (CAZymes) and selectively attacks cell wall polysaccharides, depending on the carbohydrate composition of the invaded plant tissue (Blanco-Ulate et al., 2014). Plant defense response against this pathogen are complex and involve many genes related to phytohormone signaling, including the ethylene, abscisic acid, JA, and SA pathways (Kliebenstein et al., 2005).

Glucosinolates (GSs) are sulfur-rich anionic secondary metabolites characteristic of the crucifers (the Brassicaceae family), with important biological and economic roles in plant defense and human nutrition. Currently, there are approximately 140 naturally produced GSs described in the literature (Nguyen et al., 2020). They all share a common chemical structure, consisting of a β-D-glucopyranose residue linked via a sulfur atom to a (Z)-N-hydroximinosulfate ester, as well as a variable R group. GSs are divided into three classes according to their precursor amino acid. Compounds derived from methionine, alanine, leucine, isoleucine or valine are called aliphatic GSs, those derived from phenylalanine or tyrosine are called aromatic GSs and those derived from tryptophan are called indole GSs. The various ecotypes of the model plant Arabidopsis thaliana produce about 40 different GSs of the indole and aliphatic families. GSs become biologically active only in response to tissue damage, at which point they are enzymatically cleaved by special thioglucoside glucohydrolases known as myrosinases. These enzymes hydrolyze the glucose moiety of the GS, creating an unstable aglycone that can rearrange to form nitriles, thiocyanates, isothiocyanates or other active products. To prevent damage to the plant itself, spatial compartmentalization separates myrosinases, which are mainly stored in specialized myrosin cells, from their GS substrates that are found in vacuoles throughout the plant cells (Halkier and Gershenzon, 2006). In recent years, it was demonstrated that GS metabolism is an important component of the plant defense response against fungi and other microbial pathogens (Bednarek et al., 2009; Clay et al., 2009; Buxdorf et al., 2013). The regulation of GS metabolism is a complex process involving all major plant defense hormones (i.e., SA, JA, abscisic acid, and ethylene) but also other hormones, such as gibberellic acid, brassinosteroids, and auxin are also involved (Mitreiter and Gigolashvili, 2021). Six R2R3-MYB transcription factors are known to be positive regulators of GS biosynthesis. Specifically, MYB28, MYB29, and MYB76 affect aliphatic GS (Li et al., 2013), whereas MYB34, MYB51 and MYB122 regulate indole GSs (Frerigmann and Gigolashvili, 2014a; Mitreiter and Gigolashvili, 2021).

IQD1 has also been found to be a positive regulator of GS accumulation and plant defense responses against insects (Levy et al., 2005). IQD1 is part of a family that comprises 33 IQD genes in A. thaliana, all encoding proteins possessing a distinct plant-specific domain of 67 conserved amino acids termed the IQ67 domain. The IQ67 domain is characterized by a unique and repetitive arrangement of IQ, 1-5-10 and 1-8-14 calmodulin recruitment motifs (Abel et al., 2005). IQD genes are not unique to A. thaliana, as bioinformatics and molecular tools have identified IQD genes in additional plant species, such as rice, tomato, soybean, grapevine, and others (Filiz et al., 2013; Huang et al., 2013; Feng et al., 2014; Ma et al., 2014; Cai et al., 2016; Wu et al., 2016; Yuan et al., 2019; Liu et al., 2020; Mei et al., 2021; Rehman et al., 2021).

IQD genes also play diverse roles in plants unrelated to glucosinolate synthesis or defense mechanisms. A set of microarray studies designed to identify DELLA responsive genes revealed A. thaliana IQD22 as one of several proteins involved in the early response to gibberellin (Zentella et al., 2007). SUN, the tomato IQD12 homolog was found to be a major factor controlling the elongated fruit shape of tomato fruits (Xiao et al., 2008). IQD family proteins from the cotton Gossypium hirsutum (GhIQD31 and GhIQD32) were found to induce drought and salt stress tolerance (Yang et al., 2019). A continuous body of work from the recent years has pointed to a general role for IQD proteins as microtubule-binding proteins that recruit calmodulin to subcellular compartments so as to coordinate plant development and cell shape formation (Bürstenbinder et al., 2013, 2017; Guo et al., 2020; Bao et al., 2021). Previous studies also identified the kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor in A. thaliana and demonstrated the association of IQD1 with microtubules. It was further suggested that IQD1 and related proteins provide scaffolds that facilitate cellular transport of RNA along microtubular tracks, as a mechanism to control and fine-tune gene expression and protein sorting, thus explaining the pleiotropic effects of IQD1 in many cellular pathways (Abel et al., 2013; Bürstenbinder et al., 2013). The A. thaliana IQD16 was also implicated as a microtubule-associated protein affecting cortical microtubule ordering, apical hook formation and cell expansion (Li et al., 2020). Indeed, a recent publication showed that the DUF4005 domain, frequently found in the C-terminal portion of IQD proteins, is a microtubule-binding motif (Li et al., 2021). In the current work, we sought to elucidate the mechanism of action of the A. thaliana IQD1 protein and define its involvement in hormone signaling and in basal defense against B. cinerea.

This study aimed to elucidate the molecular functions of the A. thaliana IQD1 protein in defense responses against the plant pathogen B. cinerea. Previous work with IQD1 mutants showed that the IQD1 expression levels in different A. thaliana lines correlated with steady state accumulation of glucosinolates. Moreover, these earlier efforts showed that overexpressing IQD1 has the beneficial characteristic of reducing the herbivory of generalist insects (Levy et al., 2005). Using the necrotrophic fungal pathogen B. cinerea, we sought to investigate the cellular and genetic pathways that IQD1 regulated and thus affects plant defense response. Inoculating the IQD1^OXP and iqd1-1 lines with a B. cinerea spore suspension proved the correlation between IQD1 expression levels and A. thaliana resistance to the fungal pathogen (Figure 1), as well as providing us with a simple host-pathogen system to conduct genetic screening. It was already known from our previous studies that the iqd1-1 knockout plant accumulates low levels of GS (Levy et al., 2005). In the current study, we also validated that iqd1-1 plants abnormally express several GS biosynthesis and regulation genes, as compared to WT plants (Table 2).

Information obtained from genome-wide expression profiling of iqd1-1 and WT plants following mock treatment or B. cinerea infection, helped us understand which plant metabolic processes were affected by the absence of IQD1. The latest genome model released for A. thaliana (TAIR10) contains about 27,000 protein-coding genes (Lamesch et al., 2012). We showed that approximately 3,500 genes (roughly 13% of all coding genes) were differentially expressed in the non-infected IQD1 knockout as opposed to WT plants (Supplementary Figure 1A and Supplementary Data Sheet 1). Furthermore, 70% of the genes which were downregulated in the iqd1-1 line generate products predicted to serve diverse functions, including transport, DNA repair and gene regulation. It is noteworthy that a large number of downregulated genes in the iqd1-1 line encode proteins responsible for plant defense against biotic stresses, such as cell wall remodeling proteins, signaling factors and proteins involved in resistance (Figure 2). Such a massive impairment of the plant defense apparatus is likely to explain the enhanced sensitivity of the knockout plants to insect and pathogen attacks (Levy et al., 2005; Figure 1). The ERF genes encode a large family of ethylene responsive transcription factors that regulate important biological processes related to plant growth, development and plant defense (Nakano et al., 2006; Li et al., 2019). This gene family was largely upregulated in the iqd1-1 mutant (Figure 2 and Table 1). The increased sensitivity to ethylene may explain several phenotypes displayed by this line, such as rapid growth and early development of stems and seed pods, relative to WT plants (Levy et al., 2005). As demonstrated in Figure 6 and earlier studies, ethylene can effect glucosinolate biosynthesis (Mikkelsen et al., 2003) and its signaling components EIN2 and ETO1 act downstream to IQD1 control of defense and GS accumulation.

Upon inoculation with B. cinerea, both the WT (Supplementary Figure 2) and iqd1-1 (Supplementary Figure 3) plants presented a similar basic transcriptional response, shutting down the energy-consuming photosynthesis machinery and instead concentrating efforts on fighting off the invading pathogen. The responses did, however, differ with the WT plants being able to express more defense-related genes, like those encoding germins and R-genes (Figure 2B), thus more effectively resisting fungal infection than iqd1-1 plants. As for other IQD family members, our transcriptional data demonstrated that while most genes were unaffected after B. cinerea infection, three were up-regulated in response to B. cinerea inoculation (i.e., IQD13, IQD15, and IQD27) and five are down-regulated (i.e., IQD9, IQD18, IQD19, IQD21, and IQD26). Since IQD genes products serve a wide range of cellular functions, many unrelated to defense mechanisms, it is not surprising that the levels of these genes differed as a function of the biological stress imposed in the current study.

The three plant hormones SA, JA, and ethylene play a major role in response to biotic stresses by mediating endogenous signaling that activates the expression of plant defense genes (Dong, 1998; Clarke et al., 2000; Li et al., 2019). Analysis of RNA-Seq data of iqd1-1 plants indicated that IQD1 is involved in all three major hormone defense pathways (Table 1). While we saw transcriptional changes in genes controlling all important plant hormones in WT and mutant plants, ethylene JA and ABA signaling genes were mainly upregulated in iqd1-1 plants (see above), unlike to SA metabolism genes that showed opposite behavior (Table 1). Using the IQD1^pro:GUS reporter line we showed that exogenous application of SA or Flg22 down-regulates IQD1 expression, while JA and chitin treatment led to the opposite effect, activating IQD1 expression (Figures 3A,B). Further confirmation of the link between IQD1 activity and the JA pathway came from LC/MS quantification of hormone accumulation in IQD1 mutants. We observed lower steady-state JA levels in iqd1-1 mutant plants, as compared to WT, while SA levels were significantly increased (Figure 3C). We can speculate that IQD1 represses the accumulation of SA while activating JA accumulation (Figures 3C, 9 and Table 1). It is clear from former publications that glucosinolate accumulation and metabolism are under the control of different hormone signaling pathways and several studies have demonstrated, like us, that changes in glucosinolate levels altered levels of hormone, such as JA, ET, and ABA (Mikkelsen et al., 2003; Dombrecht et al., 2007; Malitsky et al., 2008; Morant et al., 2010; Chen et al., 2011; Mitreiter and Gigolashvili, 2021). Given the results on hormone levels in the iqd1-1 mutant and expression levels of IQD1 after hormone treatment presented above, we hypothesize that the opposing effects on SA and JA levels reflected the involvement of IQD1 in the well documented synergy between the JA and SA pathways (Koornneef and Pieterse, 2008; Pieterse et al., 2012; Li et al., 2019; Figure 9).

GS metabolism is also linked to auxin homeostasis. Indole GS contribute to auxin biosynthesis via metabolic intermediates indole-3-acetaldoxime (IAOx) and indole-3-acetonitrile (IAN). IAOx thus constitutes a metabolic branch point in indole-3-acetic acid (IAA) and indole glucosinolate biosynthesis, while IAA levels can be regulated by the flux of IAOx (Bak et al., 2001; Malka and Cheng, 2017). We observed some up-regulation of auxin responsive genes in our RNA-seq study on the one hand, yet also saw the same number of down-regulated genes. On the other hand, we did not observe any auxin-related phenotype in the iqd1-1 or IQD1^OXP lines, leading us to assume that auxin levels might not change, as also seen in the of CYP79B2 overexpressing line, in which significantly elevated levels of indole GSs and IAN but normal IAA levels were seen (Mikkelsen et al., 2000; Zhao et al., 2002; Glawischnig et al., 2004). These observations raise the possibility that the IAOx pathway may not contribute to basal IAA production and to its role in regulating plant growth and development as much as other IAA biosynthesis pathways. There is some evidences that other IQD family members, such as IQD15, are connected to the auxin response (Möller et al., 2017), although IQD1 expression levels were not affected after auxin treatment in various transcriptional studies (Pufky et al., 2003; Nemhauser et al., 2004; Redman et al., 2004). Clearly, the connection between IQD1 and auxin needs to be further characterized. Based on data obtained following B. cinerea inoculation of detached leaves and GS concentration measurements by HPLC on siblings of IQD1^OXP crossed with different transgenic plants and mutants connected to hormonal pathways, we were able to investigate IQD1 integration into the three main defense hormone-signaling and response pathways. Over-expression of IQD1 did not alter resistance/sensitivity or GS levels in any of SA and ethylene pathway mutants we tested (Figures 4, 6). We thus, assume it to be more likely that IQD1 is dependent on them and that it might act upstream to hormone pathways for defense activation and GS accumulation (Figure 9). The data presented here showed that the ein2 and eto1 mutants were both more sensitive to B. cinerea from WT plants despite presenting opposite phenotypes in terms of ethylene responses. This discrepancy can be explained by the difference in GS levels: in the ein2 mutant, levels of indole GS are reduced and levels of aliphatic GS are increased, whereas the reverse was found in the eto1 mutant where levels of indole GS are increased and levels of aliphatic GS are reduced (Figure 6B). This perhaps reflects that GSs having a greater impact than ethylene on this B. cinerea isolate (Buxdorf et al., 2013). Additionally, ethylene has also been shown to induce the expression of GS biosynthetic genes and their regulators (Mikkelsen et al., 2003; Frerigmann and Gigolashvili, 2014b). Another possibility is that the elevated levels of ethylene induced B. cinerea pathogenicity (Elad, 1990; Chagué et al., 2006). Furthermore, while indole GS content in the jar1 plants was higher even than in the IQD1^OXP line, most likely due to the increase of several JA conjugates in the single mutant, as described before (Staswick and Tiryaki, 2004), the jar1 IQD1^OXP cross plants accumulated significantly less indole GSs than did the jar1 plants but similar to what was seen in WT plants (Figure 5). These results offer additional proof of the connection between IQD1 and the JA pathway. We thus, hypothesize that IQD1 acts upstream of the JA signaling pathway and is dependent on JAR1 controlled indole GS accumulation. IQD1 also controls JA accumulation by activating JA biosynthesis genes (Table 1) and is activated by JA via a positive feedback loop (for model, see Figure 9).

The extensive volume of data obtained from our RNA-Seq experiment also enabled us to investigate the properties of B. cinerea infection on iqd1-1 plants, as compared to the WT (Figure 7A). Examination of differentially expressed genes (DEGs) revealed that upon iqd1-1 infection, the fungus expressed an extensive array of CAZymes and membrane transporters, which facilitate the penetration and breakdown of plant tissues (Table 3 and Figure 8). It has been proposed that B. cinerea is able to fine tune the expression of activated CAZymes according to the carbohydrate composition of the host cell wall (Blanco-Ulate et al., 2014).

We thus hypothesize that following early penetration of the leaf tissue, the fungus can better proliferate on iqd1-1 plants since this mutant contains low levels of GSs and this can induce the expression of CAZymes that rapidly break down the physical barriers of the plant cell (Table 3 and Supplementary Figure 6). We conclude that B. cinerea infection is more aggressive on iqd1-1 plants, as the fungus takes advantage of the enhanced sensitivity of the mutant, mainly the reduction in GS levels, as also described in our previous work (Buxdorf et al., 2013).

In conclusion, the current study demonstrated that altered expression of A. thaliana IDQ1 has a profound effect on the global expression of plant genes but also those of the pathogen. Moreover, IQD1 expression correlates with GS levels, defense signaling and B. cinerea pathogenicity.

The data presented in the study are deposited in the Figshare online open access repository, accession number https://doi.org/10.6084/m9.figshare.19384043.v1.

OB and ML designed the experiments and wrote the article together. OB performed the majority of the experiments and analyzed the data, with assistance from ML. Both authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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