Exploring salicylic acid biosynthesis in Trichoderma spp. using an enhanced transformation approach.

Salicylic acid synthesized by benzoic acid 2-hydroxylase participates in the development of thermotolerance in pea plants

Cotton, as a globally significant economic crop, is intricately regulated in its growth and development by the key genes for SA (Salicylic acid) biosynthesis. In the present study, a systematic analysis of genes related to SA biosynthesis was conducted across four cotton species, leading to the identification of 70 genes. Specifically, the tetraploid species Gossypium hirsutum and G. barbadense were found to harbor 22 and 23 genes, respectively, representing a substantial expansion compared to the 12 and 13 genes identified in the diploid progenitors G. arboreum and G. raimondii. Comprehensive characterization of chromosomal localization, phylogeny, domain architecture, and promoter cis-elements revealed a uniform distribution of key genes involved in SA biosynthesis across A/D sub-genomes of tetraploids with extensive interspecific collinearity; whole-genome and segmental duplication act as the dominant drivers for the expansion of this gene family, while partial gene loss following polyploidization results in non-doubled gene copy numbers in tetraploids relative to diploids, which reflects the evolutionary selection for genomic dosage balance. The key genes for SA biosynthesis demonstrate a high degree of conservation in protein sequences, protein structures, and conserved motifs, which constitute the structural basis for the stable maintenance of their core functions in the SA biosynthesis pathway during plant evolution. This is closely related to their core function in the salicylic acid (SA) synthesis pathway and serves as the structural basis for the stable maintenance of gene functions during evolution. Analysis of cis-elements revealed that the expression of key genes involved in SA biosynthesis is governed by a complex interplay of phytohormones, stress signals, and transcription factors. Yeast one-hybrid (Y1H) assays confirmed the interaction between the GhPAL and GhICS gene and predicted candidate transcription factors, specifically the binding of GhWRKY21 to GhICS2-1 promoter and GhMYB12 to GhPAL1-2 promoter, thus elucidating their stage-specific regulatory mechanisms in cotton fiber development and reflecting their evolution. This study provides a fundamental basis for investigating the role of the SA signaling pathway in cotton development and offers support for cotton molecular breeding.

The origin and evolution of salicylic acid signaling and biosynthesis in plants

Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen.

Bioengineering and Molecular Manipulation of Salicylic Acid Signaling System to Activate Plant Immune Responses for Crop Disease Management

Pathogenic fungus Blumeria graminisforma specialistritici (B.g. tritici) is the causal agent of the devastating wheat powdery mildew disease. Identifying the key regulators governing wheat susceptibility to the B.g. tritici pathogen is essential for developing wheat varieties with improved powdery mildew resistance. In this study, we demonstrated that the wheat chromatin remodeler TaSWI3D positively regulates wheat susceptibility to B.g. tritici. Overexpression of TaSWI3D gene attenuates wheat resistance against B.g. tritici, while silencing of TaSWI3D gene potentiates wheat powdery mildew resistance. TaSWI3D protein was found to be enriched at the promoter regions of the TaSARD1 gene encoding the salicylic acid (SA) biosynthesis activator, and silencing of TaSWI3D resulted in decreased nucleosome occupancy at the TaSARD1 promoter regions. Activated TaSARD1 transcription and increased SA accumulation were observed in the TaSWI3D-silenced wheat plants. Silencing of TaSARD1 and the SA biosynthesis gene TaICS1 resulted in attenuated SA biosynthesis and decreased powdery mildew resistance in the TaSWI3D-silenced wheat plants. These findings support that the chromatin remodeler TaSWI3D maintains epigenetic suppression of the SA biosynthesis activator gene TaSARD1 and negatively regulates SA biosynthesis, thereby positively contributing to wheat powdery mildew susceptibility.

When mobile organisms face a threat, they have the options of a fight or flight as reaction. The sessile nature of plants narrows their response option down to defend themselves against the threat. Therefore, plants developed a strong innate immune system in an evolutionary context, redundant of specialized immune cells as found in animals. Besides, they are able to prime non-infected distal tissue towards a stronger immune response after pathogen attack, a phenomenon that is termed systemic acquired resistance (SAR) (Fu and Dong, 2013). Salicylic acid (SA) and N-hydroxy pipecolic acid (NHP) are small molecules and constitute two major hormones in the plant immune response. They are key molecules in basal resistance as well as to induce SAR (Delaney et al., 1994; Wildermuth et al., 2001; Chen et al., 2018; Hartmann et al., 2018; Rekhter et al., 2019b). The biosynthesis and function of SA has been intensively studied over the last decades (Wildermuth et al., 2001; Nawrath et al., 2002; Rekhter et al., 2019b; Torrens-Spence et al., 2019). In addition, the biosynthesis of NHP was unraveled recently (Chen et al., 2018; Hartmann et al., 2018). Both compounds are known to be present in a glycosylated state likely to be inactivated or stored. This is now shifting the focus towards the enzymes catalyzing the glycosylation reactions. For SA glycosylation, three UDP-dependent glycosyltransferases (UGTs) have been described: UGT74F1, UGT74F2 and UGT76B1 (Song, 2006; Dean and Delaney, 2008; von Saint Paul et al., 2011; Noutoshi et al., 2012; George Thompson et al., 2017). In terms of NHP, the NHP-O-glycoside (NHP-OGlc) was a known metabolite, without the description of a functional UGT enzyme that was able to catalyze the synthesis, prior to this thesis (Chen et al., 2018; Hartmann and Zeier, 2018). Lately, independent research groups were able to describe one of the proposed SA UGTs, UGT76B1, to be the major enzyme in the formation of NHP OGlc in Arabidopsis thaliana (Bauer et al., 2021; Cai et al., 2021; Holmes et al., 2021; Mohnike et al., 2021). In this work, the identification and functional characterization of UGT76B1 as NHP-OGlc forming enzyme is laid-out as published earlier in The Plant Cell within Mohnike et al. 2021 (Mohnike et al., 2021). The metabolite levels of NHP, SA and their respective glucosides are therein described in response to Pseudomonas syringae pv. maculicola ES4326 (P.s.m.) infection. The metabolic fate of NHP and SA in the ugt76b1 mutant was underlined by additional UV-stress experiments. In addition, we provide data about the infection phenotype against P.s.m. and Hyaloperonospora arabidopsidis Noco 2 (H.a. Noco 2), of which we deduce an enhanced resistance phenotype of the mutant. Analyzing double mutant lines of the FLAVIN-DEPENDET MONOOXYGENASE 1 (FMO1) with ugt76b1, fmo1 ugt76b1, we show that enhanced resistance and growth deficiency are FMO1-dependent, therefore, NHP-dependent. Lastly, we argue against the need of NHP-O-glucosylation for successful mobility during SAR (Chapter I). Furthermore, we used our metabolome analysis platform to search for novel, so far undescribed metabolites of NHP. A novel metabolite, which is synthesized in an infection-dependent manner, is described to be a NHP-methyl-ester (MeNHP). Its biosynthesis is shown to be AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1)- and FMO1-dependent. In addition, its retention time and tandem-mass spectrometric properties were underlined via a chemically synthesized authentic standard of MeNHP. The novel compound is synthesized in vitro by the annotated methyl transferase At4G22530 (NHPMT1). However, T-DNA insertion lines of NHPMT1, nhpmt1-1 and nhpmt1-2 are not impaired in biosynthesis of MeNHP (Chapter II). Additionally, we present a NHP and D9-labeled NHP co-infiltration experiment to identify additional in planta NHP-derivatives. Moreover, we layout results about successful repetition of earlier published work and investigated genes that remained inconclusive towards their influence and function in plant pathogen interaction mediated defense response of A. thaliana. We were able to confirm the role of ENHANCED PSEUDOMONAS SUSCEPTIBILITY1 (EPS1) on the biosynthesis of SA by the accumulation of its substrate iscochorismate-9-glutamate (IC-9-Glu) in metabolite analysis of eps1 mutant plants (Torrens-Spence et al., 2019). Furthermore, we show that the amino acid transporter LYSINE/HISTIDINE 7 (LHT7) is not solely required for NHP biosynthesis. Similarly, ABERRANT LATERAL ROOT FORMATION 5 (ALF5), ENHANCED DISEASE SUSCEPTIBLITY 5 (EDS5) and EDS5-homolog (EDS5H) are not solely required as transporters in NHP biosynthesis (Chapter III).

Nitric oxide (NO) has emerged as a key signaling molecule in plant secondary metabolite biosynthesis recently. In order to investigate the molecular basis of NO signaling in elicitor-induced secondary metabolite biosynthesis of plant cells, we determined the contents of NO, salicylic acid (SA), jasmonic acid (JA), and puerarin in Pueraria thomsonii Benth. suspension cells treated with the elicitors prepared from cell walls of Penicillium citrinum. The results showed that the fungal elicitor induced NO burst, SA accumulation and puerarin production of P. thomsonii Benth. cells. The elicitor-induced SA accumulation and puerarin production was suppressed by nitric oxide specific scavenger cPITO, indicating that NO was essential for elicitor-induced SA and puerarin biosynthesis in P. thomsonii Benth. cells. In transgenic NahG P. thomsonii Benth. cells, the fungal elicitor also induced puerarin biosynthesis, NO burst, and JA accumulation, though the SA biosynthesis was impaired. The elicitor-induced JA accumulation in transgenic cells was blocked by cPITO, which suggested that JA acted downstream of NO and its biosynthesis was controlled by NO. External application of NO via its donor sodium nitroprusside (SNP) enhanced puerarin biosynthesis in transgenic NahG P. thomsonii Benth. cells, and the NO-triggered puerarin biosynthesis was suppressed by JA inhibitors IBU and NDGA, which indicated that NO induced puerarin production through a JA-dependent signal pathway in the transgenic cells. Exogenous application of SA suppressed the elicitor-induced JA biosynthesis and reversed the inhibition of IBU and NDGA on elicitor-induced puerarin accumulation in transgenic cells, which indicated that SA inhibited JA biosynthesis in the cells and that SA might be used as a substitute for JA to mediate the elicitor- and NO-induced puerarin biosynthesis. It was, therefore, concluded that NO might mediate the elicitor-induced puerarin biosynthesis through SA- and JA-dependent signal pathways in wildtype P. thomsonii Benth. cells and transgenic NahG cells respectively.

Salicylic acid (SA) biosynthesis, the expression of SA-related genes and the effect of SA on the Arabidopsis-Plasmodiophora brassicae interaction were examined. Biochemical analyses revealed that, in P. brassicae-infected Arabidopsis, the majority of SA is synthesized from chorismate. Real-time monitored expression of a gene for isochorismate synthase was induced on infection. SA can be modified after accumulation, either by methylation, improving its mobility, or by glycosylation, as one possible reaction for inactivation. Quantitative reverse transcription-polymerase chain reaction (qPCR) confirmed the induction of an SA methyltransferase gene, whereas SA glucosyltransferase expression was not changed after infection. Col-0 wild-type (wt) did not provide a visible phenotypic resistance response, whereas the Arabidopsis mutant dnd1, which constitutively activates the immune system, showed reduced gall scores. As dnd1 showed control of the pathogen, exogenous SA was applied to Arabidopsis in order to test whether it could suppress clubroot. In wt, sid2 (SA biosynthesis), NahG (SA-deficient) and npr1 (SA signalling-impaired) mutants, SA treatment did not alter the gall score, but positively affected the shoot weight. This suggests that SA alone is not sufficient for Arabidopsis resistance against P. brassicae. Semi-quantitative PCR revealed that wt, cpr1, dnd1 and sid2 showed elevated PR-1 expression on P. brassicae and SA + P. brassicae inoculation at 2 and 3 weeks post-inoculation (wpi), whereas NahG and npr1 showed no expression. This work contributes to the understanding of SA involvement in the Arabidopsis-P. brassicae interaction.

The fungal pathogen Blumeria graminis forma specialis tritici (B.g. tritici) infects bread wheat (Triticum aestivum L.) to cause wheat powdery mildew disease. Elucidating the molecular mechanism underlying wheat susceptibility to the pathogenic fungus B.g. tritici could facilitate wheat genetic improvement. In this study, we identified the wheat TaSWI3B gene as a novel Susceptibility gene positively regulating wheat susceptibility to B.g. tritici. The TaSWI3B gene encodes the SWI3B subunit of the SWI/SNF chromatin remodeling complex. The overexpression of the TaSWI3B gene enhances wheat powdery mildew susceptibility, whereas TaSWI3B silencing results in attenuated wheat powdery mildew susceptibility. Importantly, we found that TaSWI3B could be enriched at the promoter regions of the salicylic acid (SA) biosynthesis activator gene TaSARD1, facilitating nucleosome occupancy and thereby suppressing TaSARD1 transcription and inhibiting SA biosynthesis. Silencing of TaSARD1 and TaICS1 encoding a key enzyme in SA biosynthesis could attenuate the SA biosynthesis and powdery mildew resistance potentiated by knockdown of TaSWI3B expression. Collectively, these results suggest that the SWI3B subunit of the wheat SWI/SNF chromatin remodeling complex negatively regulates SA biosynthesis by suppressing TaSARD1 transcription at the epigenetic level and thus facilitates wheat powdery mildew susceptibility.

Immune signaling networks must be tunable to alleviate fitness costs associated with immunity and, at the same time, robust against pathogen interferences. How these properties mechanistically emerge in plant immune signaling networks is poorly understood. Here, we discovered a molecular mechanism by which the model plant species Arabidopsis thaliana achieves robust and tunable immunity triggered by the microbe-associated molecular pattern, flg22. Salicylic acid (SA) is a major plant immune signal molecule. Another signal molecule jasmonate (JA) induced expression of a gene essential for SA accumulation, EDS5 Paradoxically, JA inhibited expression of PAD4, a positive regulator of EDS5 expression. This incoherent type-4 feed-forward loop (I4-FFL) enabled JA to mitigate SA accumulation in the intact network but to support it under perturbation of PAD4, thereby minimizing the negative impact of SA on fitness as well as conferring robust SA-mediated immunity. We also present evidence for evolutionary conservation of these gene regulations in the family Brassicaceae Our results highlight an I4-FFL that simultaneously provides the immune network with robustness and tunability in A.thaliana and possibly in its relatives.

Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-beta-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance.

Wheat powdery mildew disease caused by the obligate biotrophic fungal pathogen Blumeria graminis forma specialis tritici (B.g. tritici) seriously threatens global wheat production. Although improved powdery mildew resistance is an aim in wheat breeding, the regulatory mechanism underlying the wheat-B.g. tritici interaction remains poorly understood. In this study, the wheat chromatin remodeling protein TaSWP73 was identified as a negative regulator of post-penetration resistance against B.g. tritici. The transient overexpression of TaSWP73 attenuates wheat post-penetration resistance against B.g. tritici, while the silencing of TaSWP73 potentiates salicylic acid (SA) biosynthesis and activates post-penetration resistance against B.g. tritici. Importantly, chromatin in the promoter regions of TaSARD1, an activator gene of SA biosynthesis, is marked by high nucleosome occupancy in the TaSWP73-silenced wheat leaves. The silencing of TaSARD1 could suppress SA biosynthesis and attenuate post-penetration resistance against B.g. tritici with a lack of TaSWP73. In addition, TaICS1 was characterized as an essential component of wheat SA biosynthetic machinery. Potentiated SA biosynthesis and increased post-penetration resistance against B.g. tritici with a lack of TaSWP73 could be suppressed by the silencing of TaICS1 expression. These results collectively support the hypothesis that the wheat chromatin remodeling protein TaSWP73 contributes to the compatible wheat-powdery mildew interaction presumably via the suppression of the TaSARD1-TaICS1-SA pathway.

Temporal dynamics of N-hydroxypipecolic acid and salicylic acid pathways in the disease response to powdery mildew in wheat

Salicylic Acid Signaling in Plant Innate Immunity