The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced innate immunity.

The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced innate immunity. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 31 January 2024 V1 Latest version Share on The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced innate immunity. Authors : Ching-Han Chang , Wu-Guei Wang , Pei-Yu Su , Yu-Shuo Chen , Tri-Phuong Nguyen , Jian Xu , Masaru Ohme-Takagi , Tetsuro Mimura , and Hao-Jen Huang 0000-0002-7459-162X [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.170670996.69208775/v1 289 views 111 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract ​​ Microbial volatile organic compounds (mVCs) are a part of a collection of microbial secondary metabolites with biological effects on all living organisms. mVCs could function as gaseous modulators of plant growth and plant health. In this study, the defense events induced by plant-deleterious mVCs were investigated. E . aerogenes VCs lead to growth inhibition and plant defense responses such as callose deposition, and ROS accumulation in Arabidopsis thaliana . Data from transcriptional analysis suggests that genes involved in the hypoxia response pathway were enriched in the short exposure up-regulated genes, E . aerogenes VCs induced high transactivation of defense, immune, and metabolic processes after long exposure. In addition, the transcript abundance of the genes involved in the synthetic pathways of antimicrobial metabolites camalexin and coumarin was enhanced after the E . aerogenes VCs exposure. MKK1 and MKK3 were identified as the regulators of the camalexin biosynthesis expression and E . aerogenes VCs-induced callose deposition. The transactivation activity of the coumarin biosynthesis pathway was only regulated by MKK3. Collectively, these studies provide molecular insights into immune responses by plant-deleterious mVCs. Title: The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced innate immunity. Authors: Ching-Han Chang 1 , Wu-Guei Wang 2 , Pei-Yu Su 3 , Yu-Shuo Chen 2 , Tri-Phuong Nguyen 3 , Jian Xu 4 , Masaru Ohme-Takagi 2 , Tetsuro Mimura 1 , Hao-Jen Huang 1,2,3* Affiliation: 1 Translational Agricultural Sciences, NCKU-AS, Tainan, Taiwan 2 Institute of Tropical Plant Sciences and Microbiology, NCKU, Tainan, Taiwan 3 Department of Life Sciences, NCKU, Tainan, Taiwan 4 Department of Plant Systems Physiology, Radboud University, Nijmegen, The Netherlands *Correspondence: Hao-Jen Huang Tel: 886-6-2757575 ext. 58104 Email: [email protected] Hao-Jen Huang ORCID https://orcid.org/0000-0002-7459-162X Running head: AtMKK1 and AtMKK3 regulate plant-deleterious microbial volatile compounds-induced innate immunity Abstract ​​ Microbial volatile organic compounds (mVCs) are a part of a collection of microbial secondary metabolites with biological effects on all living organisms. mVCs could function as gaseous modulators of plant growth and plant health. In this study, the defense events induced by plant-deleterious mVCs were investigated. E . aerogenes VCs lead to growth inhibition and plant defense responses such as callose deposition, and ROS accumulation in Arabidopsis thaliana . Data from transcriptional analysis suggests that genes involved in the hypoxia response pathway were enriched in the short exposure up-regulated genes, E . aerogenes VCs induced high transactivation of defense, immune, and metabolic processes after long exposure. In addition, the transcript abundance of the genes involved in the synthetic pathways of antimicrobial metabolites camalexin and coumarin was enhanced after the E . aerogenes VCs exposure. MKK1 and MKK3 were identified as the regulators of the camalexin biosynthesis expression and E . aerogenes VCs-induced callose deposition. The transactivation activity of the coumarin biosynthesis pathway was only regulated by MKK3. Collectively, these studies provide molecular insights into immune responses by plant-deleterious mVCs. Keywords: ​​ Enterobacter aerogenes , mVCs, metabolism, immune, AtMKK1, AtMKK3 1. Introduction In nature, plants provide habitats for a variety of microbes, insects and other animals and play essential roles in the ecosystems as the producers. To survive, plants have intricate mechanisms which equip them with effective defense responses against biotic stimuli. The plant-microbe interactions have diverse effects on plant growth, development, plant health and responses toward the biotic and abiotic stimuli. Based on its effects on plant performance, microbes could be classified into beneficial and detrimental microbes (Bowen and Rovira 1999). The beneficial, infective consist of the symbiotic microbes which improve plant’s nutrient uptake, growth or health; the beneficial, non-infective microbes are composed of plant growth promoting microbes (PGPMs) (Bowen and Rovira 1999). PGPMs also lead to growth promotion, enhancement of plant resistance against biotic stimuli, and confer plant tolerance under abiotic stresses (Gururani et al ., 2013; Pii et al . 2015; Souza, Ambrosini & Passaglia 2015; Vurukonda, Vardharajula, Shrivastava & SkZ 2016; Barnawal et al . 2017). The detrimental, infective microbes consist of the pathogens which cause plant growth inhibition and disease symptoms; the detrimental, non-infective microbes are composed of deleterious microbes (Bowen and Rovira 1999). The plant-deleterious microbes do not cause disease symptoms or build the parasitism but would suppress plant growth. Recently, Finkel et al . (2020) showed that 34.2 % of the root-associated microbes could lead to growth inhibition. When inoculating the root-associated microbiota with Arabidopsis , the primary roots growth exhibits a significant decrease comparing to the seedlings without microbiota inoculation (Finkel et al . 2020). To be able to monitor the presence of microbes, plants have evolved the sophisticated signaling network to perceive and modulate their response toward the biotic stimuli. Plants are able to perceive microbes through recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) such as flg22, chitins and EF-Tu via pattern recognition receptors (PRRs) (Gómez-Gómez & Boller 2000; Miya et al . 2007; Zipfel et al . 2006; Albert, Hua, Nürnberger, Pruitt & Zhang 2020). Other than microbial elicitors, wounding-induced damage-associated molecular patterns (DAMPs) could also lead to the activation of immune responses (Bacete, Mélida, Miedes & Molina 2018; Li, Wang & Mou 2020; Tanaka & Heil 2021). DAMPs, including phytocytokine, are plant-derived immunogenic factors which could be recognized by PRR complexes (Tanaka & Heil 2021). The activation of the PRR complex will lead to the induction of downstream signaling components such as MAPK cascades, ROS burst as well as salicylic acid (SA)-, jasmonic acid (JA)- and ethylene (ET)-mediated defenses (Zhou & Zhang 2020; Yuan, Ngou, Ding & Xin 2021). The recognition of MAMPs, DAMPs and phytocytokines by PRR complexes function in the regulation of immune responses (Hou, Liu & He 2021). Plant defense responses could be classified into tissue defense and molecular defense. The tissue defense responses include stomatal closure and callose deposition (Zhou, Sun & Xing 2013). The molecular defenses include the secretion of antimicrobial substrates such as antimicrobial peptides, phytoalexins and apoplastic proteases (Ebel 1986; Ahuja, Kissen & Bones 2012; Campos, Souza, Oliveira, Dias & Franco 2018; Godson & Hoorn 2021). The MAMP-mediated immunity allows plants to defend against pathogens rapidly. To repress the MAMP-mediated immunity, the pathogens would secret effector proteins into plant cells to interfere with MAMP-mediated signalings. Effector-mediated immunity is initiated with the recognition of effector proteins via nucleotide-binding leucine-rich repeat receptors (NB-LRRs) in the cytoplasm of plant cells (Peng, Wersch & Zhang 2018). The detection of effectors also leads to the induction of ROS burst, MAPK cascades and SA-, JA-, and ET-mediated defenses (Zhou & Zhang 2020; Yuan et al . 2021). The MAMP- and effector-signaling co-regulated components are activated at different time points and amplitudes (Mine et al . 2018; Yuan et al . 2021). Plant systemic immunity is classified into the systemic acquired resistance (SAR) and induced systemic resistance (ISR) based on the inducing microbes. SAR is triggered by pathogens while ISR is activated by plant-beneficial microbes (Pieterse et al . 2014). The activation of Pathogen-related gene 1 (PR1) is commonly characterized as SAR activation (Ryals et al. 1996; Loon, Rep & Pieterse 2006). Other than SAR, the beneficial microbes induced resistance is defined as the induced systemic resistance (ISR) (Pieterse et al . 2014). MYB72 is the central regulator of ISR activation. It was indicated that myb72 mutant fails to activate ISR when colonized by beneficial microbes (Van der Ent et al. 2008). An iron-deficiency response is characterized as the other ISR-activated response (Zamioudis, Hanson & Pieterse 2014). Evidence shows that numerous MYB72 modulated transcriptional changes are associated with the iron-deficiency response which could be activated by either the beneficial microbes or the microbial volatiles of plant growth-promoting microbes (Zhang et al . 2007a; Zamioudis et al . 2014). In addition to MAMPs and effectors, plants are known to detect microbial volatiles. mVCs are small molecules with low boiling points, high vapor pressure, and high lipophilicity which are derived from different metabolic pathways in microbes. mVCs such as alkanes, alkenes, alcohols, esters, ketones, sulfur compounds, and terpenoids have been identified which consist of microbial volatilome (Kanchiswamy, Malnoy & Maffei 2015). Due to its chemical characteristics, mVCs are usually emitted in the gaseous state and serve as the cross-talk mediator in organismal interactions (Kanchiswamy et al. 2015). Previous evidence has revealed that plant growth (Ryu et al. 2003; Park, Dutta, Ann, Raaijmakers & Park 2015; Nieto-Jacobo et al. 2017; Wenke, Kopka, Schwachtje, Van Dongen & Piechulla 2019), stress response (Jalali, Zafari & Salari 2017; Sukkasem, Kurniawan, Kao & Chuang 2018), plant metabolite production (Nieto-Jacobo et al. 2017; Wenke et al. 2019), and plant resistance against pathogens (Ryu et al. 2004; Han et al. 2006; Huang et al. 2012; Lee et al. 2012; D’Alessandro et al. 2014) could be manipulated through microbial volatiles in plants. In addition, dual effects of microbial volatiles on plant growth based on different dose have been investigated (Song et al . 2022). Since 2003, the effects that volatiles of growth-promoting rhizobacteria promote growth in Arabidopsis have been proven (Ryu et al . 2003). Thus far, there are many studies analyzing the effects of plant growth-promoting volatiles. Volatiles emitted by beneficial bacteria Bacillus subtilis GB03 could promote plant growth. Evidence demonstrates that volatiles of B . subtilis GB03 enhance photosynthetic capacity through manipulating sugar metabolism and ABA signaling in Arabidopsis (Zhang et al . 2008). Recent evidence further demonstrates that VCs of Trichoderma atroviride promote the Arabidopsis growth by targeting sucrose metabolism and transport (Esparza-Reynoso et al . 2021). Most of the studies focus on the effects of plant growth-promoting volatiles. Only a few studies have explored the underlying mechanisms caused by plant growth-inhibiting volatiles. The deleterious volatiles of Serratia plymuthica and Stenotrophomonas maltophilia lead to growth inhibition and ROS accumulation in Arabidopsis thaliana (Wenke et al . 2012). WRKY18 has been identified as the growth regulator in A . thaliana (Wenke et al . 2012). Several regulation components and pathways activated by plant growth-promoting volatiles have been identified in previous studies; however, the molecular pathways which respond to the plant-deleterious mVCs are still unclear. In our previous study, we demonstrated that mpk1-mediated cell wall integrity (CWI) signaling is an important regulator to cellular protection against Enterobacter aerogenes VCs in yeast (Wu et al . 2019). Here, we found growth inhibition in A . thaliana after long exposure to E . aerogenes VCs. Tissue defense response callose deposition was induced after the exposure of E . aerogenes VCs. The analysis of transcriptional changes demonstrated that genes involved in the hypoxia pathway, ISR-like responses, and antimicrobial substrates biosynthesis were largely transactivated after E . aerogenes VCs exposure. E . aerogenes VCs induced both the wounding response and MAMPs signaling. Besides, we also identified that MKK1/3 serve as the regulators of the defense response under E . aerogenes VCs stress. This study thus demonstrates a previously uncovered molecular pathway between plant-deleterious mVCs and the defense response in plants. 2. Materials and Methods 2.1 Plant growth conditions Seeds of A . thaliana ecotype Columbia-0 (Col-0), mkk1 and mkk3 T-DNA insertion mutant lines (provided from Dr. Heribert Hirt), DR5::GFP and PINs::PINs-GFP (provided from Dr. Jian Xu) were surface sterilized by soaking the seeds in 1.6 % NaClO (w/v) containing 0.067 % tween20 (v/v) for 8 mins. Then the seeds were rinsed in sterile distilled water 5 times and vernalized for 2 days at 4 ℃ in dark conditions. After being vernalized, A . thaliana seeds were grown on ¼ Murashige and Skoog (MS) medium in a 22 ℃ growth chamber with a 16-h photoperiod for 5-6 days. 2.2 Bacterial growth and plant co-culture experiments E . aerogenes (soil isolate, BCRC number 81142) was culture in Lysogeny broth (LB) medium for routine maintenance and plant co-culture experiments at 28 °C in dark condition. For plant mVCs treatment, 100 µl of E . aerogenes suspensions was applied to the LB medium and cultured for 18 hr. The density of all cell suspensions was measured by the spectrophotometer (U-2800A, Hitachi Technologies, Tokyo, Japan) at a wavelength of 600 nm. 6 days old seedlings grown on ¼ MS medium and LB medium with or without the inoculation of E . aerogenes were transferred to a sealed co-culture equipment (Figure 1a) in a 22 °C growth chamber with a 16-h photoperiod for further experiments. 2.3 Analysis of auxin response The samples were embedded in 50 % glycerol and visualized under Leica DMLB fluorescence microscopy with a GFP filter (excitation at 450-490 nm; emission at 500-550 nm). 2.4 Analysis of Callose Deposition Arabidopsis was incubated in 1:3 acetic acid/ethanol overnight and then washed in 150 mM K 2 HPO 4 , pH 9.5 for 30 mins. The seedlings were then stained in 150 mM K 2 HPO 4 containing 0.01 % aniline blue in dark for 30 mins. The samples were embedded in 50 % glycerol for further observation. The callose deposition was visualized under Leica DMLB fluorescence microscopy with a DAPI filter (excitation at 340-380 nm; emission at 425 nm). 2.5 RNA extraction The total RNA was extracted from the whole seedling with RNeasy Plant Mini kit (Qiagen, Hilden, German). DNase I recombinant (Roche, Basel, Switzerland) was used to catalyze DNA degradation. The RNA was purified with the RNeasy MInElute Cleanup kit (Qiagen, Hilden, Germany). The RNA was quantified by NanoDropTM 2000c Spectrophotometers (Thermo Scientific). 2.6 RNA-seq analysis The total RNA of A . thaliana whole seedlings with or without exposure to E . aerogenes VCs at 3, or 24hr was used for the RNA-seq library preparation. Each treatment had 2 biological replicates. RNA-seq libraries were constructed for the samples with or without E . aerogenes VCs for 3 hr by following the construction of Illumina NovaSeq platform which generates 150bp paired-end reads. RNA-seq libraries were constructed for the samples with or without E . aerogenes VCs for 24 hr by following the construction of Illumina HiSeq platform which generates 150bp paired-end reads. The Illumina sequencing were performed by Genewiz, Inc (Plainfield, NJ, USA). Adaptor sequences and poor-quality bases were removed by Trimmomatic v0.36 (Bolger, Lohse & Usadel 2014) with a quality score threshold set at 30. Reads were mapped to A. thaliana genome (TAIR10) by TopHat v2.1.1. Transcript assemblies were performed by Cufflinks v2.2.1 (Trapnell et al . 2012). Transcript abundance estimation and tests for differential expression were performed by Cuffdiff v2.2.1 (Trapnell et al . 2012). Genes with FPKM value = 0 were excluded in the following analysis. A gene was identified as the differentially expressed gene if it showed a | log2(FC) | >= 1 in expression (p < 0.05). Network construction of the 3hr E . aerogenes VCs up-regulated genes was performed by the web-based tool STRING with the default setting (Franceschini et al . 2012). Gene Ontology enrichment analysis was performed by BiNGO v3.0.3. The significance analysis of overrepresented categories was performed by a Hypergeometric test. False discovery rate was performed by the Benjamini & Hochberg method. 2.7 RT-qPCR 1000 ng RNA of each sample was used for Complementary DNA (cDNA) synthesis by ImProm-IITM Reverse Transcriptase (Promega). RT-qPCR was performed using GoTaq®qPCRMasterMix (Promega) on StepOnePlus Real-Time PCR Systems (Applied Biosystems). The primer sequences are listed in Table S4. Actin 8 was used as a reference in all experiments. Relative gene expression was determined by the following formula (Schmittgen & Livak 2008), Fold change (FC) = 2-𝚫𝚫CT=[(CTgene-CTreference)sampletreated-(CTgene-CTreference)sampeuntreated] 3. Results 3.1 E . aerogenes VCs induce plant defense responses Growth effects of E . aerogenes VCs treatment for 24 hr on A . thaliana are shown in Figure 1. Significant growth inhibitory effect of E . aerogenes VCs could be observed after E . aerogenes VCs exposure for 24 hr ( Figure 1b, c ). The results indicate that E . aerogenes VCs inhibit Arabidopsis growth. ROS plays an essential role in plant biotic and abiotic stress response and immune response signaling (Baxter, Mittler & Suzuki 2014; Qi, Wang, Gong & Zhou 2017). Thus, ROS production could be used as an indicator of stress responses. To examine whether E . aerogenes VCs lead to plant stress responses in Arabidopsis , ROS production was analyzed after 3 hr E . aerogenes VCs exposure. The result shows that ROS production was significantly induced after short exposure of E . aerogenes VCs ( Figure 1d ). To examine whether the E . aerogenes VCs activate tissue defense response in A . thaliana , the aniline blue was used for the quantification of callose deposits. The deposition of callose was measured after E . aerogenes VCs exposure for 48 hr in roots. The result shows that callose deposition was triggered at the elongation zone after the exposure to E . aerogenes VCs for 48 hr ( Figure 1f ). The results suggest that plant tissue defense responses could be induced not only via MAMPs or effectors recognition but also by mVCs. 3.2 E . aerogenes VCs modulate plant growth via auxin. It has been proved that microbe-induced root growth inhibition requires auxin signals (Finkel et al . 2020). To investigate whether auxin response is involved in the E . aerogenes VCs-induced root growth inhibition, auxin expression was examined. The data demonstrates that the DR5 expression was significantly decreased after E . aerogenes VCs exposure ( Figure 2a ). In addition, the expression of auxin transporter PIN3 and PIN4 were significantly decreased after E . aerogenes VCs exposure ( Figure 2b, Figure S1 ). The result suggests that auxin signals contribute to the E . aerogenes VCs induced root inhibition. 3.3 E . aerogenes VCs induce immune transcriptome changes While plants are challenged by a variety of environmental stimuli, the plant cells perceive and pass on signals by the signal transduction pathway. The signal transductions regulate plant transcriptional reprogramming in response to environmental stimuli. Thus, to identify the features of transcriptional changes in A . thaliana under E . aerogenes VCs exposure, RNA-seq analysis was performed to uncover the transcriptional changes at the E . aerogenes VCs exposure for 3, 24 hr in A . thaliana . After exposure to E . aerogenes VCs for 3 hr, a total of 73 differentially expressed genes were identified. Among the 73 genes, 33 genes were up-regulated, while 40 genes were down-regulated. In total, 2135 genes were differentially expressed at late exposure to E . aerogenes VCs. Among the 24 hr E . aerogenes VCs exposure differentially expressed genes, 1304 genes were up-regulated, and 831 genes were down-regulated ( Figure 3a ). Thus, the 3 hr E . aerogenes VCs treatment is defined as the short exposure, and 24 hr E . aerogenes VCs treatment is definded as the late exposure. The transcriptional changes induced after short exposure to E . aerogenes VCs indicate that plant transcriptome responds to mVCs stimuli rapidly. Gene ontology (GO) term enrichment analysis reveals that up-regulated genes after short E . aerogenes VCs exposure are enriched in the category related to hypoxia response ( Figure 3b ). The results demonstrate that the hypoxia pathways are involved in the E . aerogenes VCs induced signaling and response at the early stages. Functions of up-regulated genes identified after long E . aerogenes VCs exposure are mostly related to stress response, immune response, signaling, phosphorylation, and metabolism regulation ( Figure 3c ). To identify the hub genes of E . aerogenes VCs inducible genes, network component analysis was performed in STRING database. The data demonstrates that 9 of 10 hub genes are involved in plant immune response ( Figure 3d ). The other gene, STZ functions in abiotic stress regulation ( Figure 3d ). RRs mediate both MAMPs and wound signals. The data in this study demonstrates that E . aerogenes VCs exposure enhances the transcript abundance of a myriad of PRRs ( Table 1, Table S2 ). Among the 11 E . aerogenes VCs inducible PRRs, 4 PRRs are the wounding inducible components (Zhou et al . 2020) ( Table 1 ). To confirm that E . aerogenes VCs induce plant immune responses, the expression of immunity marker genes, WRKY33, FRK1 were investigated. WRKY33 is a transcription factor, which responds to MAMP flg22, and is involved in plant immunity regulation (Birkenbihl et al. 2012). FRK1 (flg22-induced receptor-like kinase 1) is a MTI-activated gene that regulates early defense gene expression (Asai et al. 2002; Porter, Shimono, Tian & Day 2012). The transcript abundance of WRKY33 and FRK1 was examined by RT-qPCR. The result indicates that WRKY33 and FRK1 would be significantly up-regulated by long E . aerogenes VCs exposure ( Figure 3e ). 3.4 E . aerogenes VCs induced similar immune transcriptional changes to MAMP flg22 Comparing E . aerogenes VCs inducible genes to the flg22 and wounding inducible genes (Ikeuchi et al . 2017; Stringlis et al . 2018a), the data in this study indicates that 69.2 % of E . aerogenes VCs inducible genes were the flg22 inducible genes ( Figure 4a ). 53 % of E . aerogenes VCs inducible genes were up-regulated by wounding ( Figure 4a ). To gain insight into the immunity related gene expression pattern in response to E . aerogenes VCs, flg22 and wounding, the gene expression of phytocytokines, MAPK cascades, defense related transcription factor (WRKYs and ERFs) and phytoalexin biosynthesis in response to E . aerogenes VCs, flg22 and wounding were examined. The results show that E . aerogenes VCs inducible immune genes show similar expression pattern to flg22 treatment ( Figure 4b ). 3.5 The involvement of MKK1 and MKK3 in the regulation of E . aerogenes VCs-induced phytoalexin biosynthesis and callose deposition The secretion of antimicrobial substrates in the apoplastic space forms the chemical barrier outside of plant cells. In Arabidopsis , the identified antimicrobial peptides include α/β-thionins, defensins, lipid transfer proteins, snakins, and 2S albumins (Campos et al . 2018). To figure out whether the antimicrobial substrates are involved in the E . aerogenes VCs activating any defense, the transcript abundance of the antimicrobial peptides and phytoalexin biosynthesis genes was examined. The data in this study indicates that a total of 9 antimicrobial peptides were up-regulated by E . aerogenes VCs ( Table 2 ). Among the 9 E . aerogenes VCs inducible antimicrobial peptides, defensins contributed to the largest proportion (4/9= 44 %) ( Table 2 ). The antimicrobial chemicals include indolic glucosinolates, triterpenes, phytoalexins camalexin, and coumarin. The data in this research demonstrates that the transcript abundance of most of the genes involved in camalexin and coumarin biosynthesis was enhanced after E . aerogenes VCs exposure ( Figure 4b ). It has been suggested that MKK1/3 cascades are involved in the up-regulation of the defensin PDF1.2 expression by the defense-related hormone JA and the regulation of immune response (Penninckx, Thomma, Buchala, Métraux & Broekaert 1998; Dóczi et al. 2007; Takahashi et al. 2007; Qiu et al. 2008​). To figure out whether MKK1/3 cascades also contribute to the transactivation of PDF1.2 by E . aerogenes VCs, the mRNA abundance of PDF1.2 in mkk1 and mkk3 mutants were examined. The results show that the transcript abundance of PDF1.2 was significantly enhanced after long exposure to E . aerogenes VCs but increased less in mkk1 and mkk3 mutants ( Figure 5a ). To examine whether MKK1/3 cascades are also the regulators of E . aerogenes VCs-induced phytoalexin biosynthesis gene transactivation, the expressions of camalexin and coumarin biosynthesis pathway genes in mkk1 and mkk3 mutants were examined. The result demonstrates that the CYP71A13 and PAD3 which encode the enzymes that catalyze the camalexin production had less transcript abundance in both mkk1 and mkk3 mutants than in the wild type after long E . aerogenes VCs exposure ( Figure 5b ). MYB72 and F6’H1 encode the proteins that catalyze the coumarin biosynthesis. Transcript abundance of MYB72 and F6’H1 was enhanced after the long E . aerogenes VCs exposure in the wild type but increased less in the mkk3 mutant ( Figure 5c ). The result suggests that MKK1 and MKK3 are involved in the transactivation regulation of camalexin biosynthesis genes. Moreover, only MKK3 is involved in the coumarin biosynthesis regulation. To examine whether MKK1 and MKK3 are required for the E . aerogenes VCs-induced tissue defense, the density of callose deposits in mkk1 and mkk3 mutants was analyzed. The result shows that the callose deposits were significantly less activates in mkk1 and mkk3 mutants ( Figure 6 ), which suggests the involvement of MKK1 and MKK3 in E . aerogenes VCs induced tissue defense. 4. Discussion 4.1 The underlying mechanisms of growth modulation by E . aerogenes VCs It has been demonstrate that flg22 inhibits plant growth via its PRR receptor FLS2 (Gómez-Gómez & Boller 2000). The data in Stringlis et al . (2018a) indicates that flg22 up-regulates defense genes and down-regulates growth-related genes. In the flg22 treated plant, the expression of the DR5 was shown to be repressed, which indicates the involvement of auxin in the flg22-induced growth inhibition (Pieterse et al . 2021). Collectively, flg22-FLS2 signaling could not only modulate immune responses, but also result in the plant growth inhibition. Moreover, auxin involves in the flg22-induced growth inhibition. The data in this study demonstrates that auxin accumulation and transport were reduced in response to the E . aerogenes VCs treatment. The comparison of the flg22 and E . aerogenes VCs inducible genes shows that a large proportion of E . aerogenes VCs inducible genes also respond to flg22 treatment. Besides, gene expression profiles of immune genes induced by E . aerogenes VCs are similar to the gene expression pattern in response to flg22. The result suggests that E . aerogenes VCs induced MAMPs signaling might contribute to the plant growth inhibition via the regulation of auxin accumulation and transport. 4.2 Hypoxia signaling might be the upstream regulatory mechanism that drives mVCs-induced immune response Plants have flexible ability to deal with different levels of low oxygen condition, such as the hypoxia, which refer to the small decrease of O 2 concentration, or anoxia, which refer to the absence of O 2 (Bailey-Serres & Voesenek 2008). Numerous factors could lead to the low oxygen condition which include submergence, pathogen infection, and the diffusion resistance in plant tissue (Bailey-Serres & Voesenek 2008; Armstrong, Webb, Darwent & Beckett 2009; Zhao et al. 2012; Kim, Jang & Park 2018; Vicente et al. 2019). The direct evidence of pathogenic induced local hypoxia at the infection site was provided by (Valeri et al . 2021). Moreover, the gene ontology enrichment analysis of MAMPs flg22 and chitin induced differentially expressed genes indicated that genes involved in the hypoxia process respond to the MAMPs treatment (Stringlis et al . 2018a; Teixeira et al . 2021). Data in Hsu et al . (2013) showed that submergence enhances the transcript abundance of immunity responsive genes and disease resistance via the regulation of WRKY22. In our study, we found that numberous hypoxia response genes are up-regulated after the short E . aerogenes VCs exposure. After long E . aerogenes VCs exposure, the immune transcriptomic changes and callose deposition are activate. To investigate whether WRKY22 is respond to E . aerogenes VCs exposure, the gene expression of WRKY22 was examined. The data indicates that WRKY22 was transactivated after E . aerogenes VCs exposure. The result suggests that WRKY22 might contribute to the up-regulation of E . aerogenes VCs responsive immunity components. Ethanol is the end product of alcoholic fermentation which was found to enhance the stress tolerance in Arabidopsis (Nguyen et al. 2017; Sako, Sunaoshi, Tanaka, Matsui & Seki 2018). To characterize short E . aerogenes VCs exposure up-regulated genes, the comparisons among microbial elicitors inducible genes (Stringlis et al . 2018a), hypoxia-responsive genes (Mustroph et al . 2010), low oxygen responsive genes (Dongen et al . 2009) and short E . aerogenes VCs inducible genes were performed. 51.5 % of short E . aerogenes VCs inducible genes were also inducible genes of hypoxia and low oxygen responsive genes ( Figure S2 ). The results indicate that short E . aerogenes VCs exposure might induce hypoxia signaling. The transactivation of alcoholic fermentation genes after the short E . aerogenes VCs exposure suggests that the alcoholic fermentation process might play a role in the E . aerogenes VCs induced responses. The data in Hann et al . (2014) demonstrated that ethanol modulates the activation of DAMP- and PAMP-responsive MAPKs and ROS burst. The result suggests that ethanol could function as a mediator of plant immunity induction. The data in this study shows that hypoxia pathway genes were up-regulated after the short stage of E . aerogenes VCs exposure. After the long E . aerogenes VCs exposure, a myriad of MTI-responsive components were transactivated. The result suggests that the transactivation of hypoxia pathway genes might contribute to the up-regulation of MTI-responsive components after the long E . aerogenes VCs exposure ( Figure 7 ). 4.3 Modulation of plant defense system in response to mVCs There is a myriad of effects caused by mVCs that have been identified. Although there are numerous studies trying to uncover the mVCs-induced signaling regulation, the underlying regulatory process in plants is still an incomplete puzzle. Among the studies, analyses of possible growth-promoting mVCs induced regulatory mechanisms contribute a large proportion to the studies of the plant-mVCs interaction (Kishimoto, Matsui, Ozawa & Takabayashi 2005; Li et al. 2011, 2018; Hao et al. 2016; Cordovez et al. 2017, 2018; Garcı́a-Gómez et al. 2020; Morcillo et al. 2020; Sun et al. 2020; Weisskopf, Schulz & Garbeva 2021). Studies related to the underlying regulatory mechanisms induced by growth-inhibiting mVCs in plants are still rare (Wenke et al . 2012, 2019). The data in this study demonstrates that not only pathogens but microbial volatiles emitted by deleterious bacteria would lead to ROS production, the deposition of callose, growth inhibition, the transactivation of MTI maker genes and biosynthesis genes involved in antimicrobial substrates and phytocytokines and ISR regulating genes. The induction of ROS production and callose deposition are the immune responses mediated by MTI signaling (Melotto, Underwood, Koczan, Nomura & He 2006 ​​; Yi, Shirasu, Moon, Lee & Kwon 2014). Recently, Zhou et al . (2020) showed that the activation of the immune response requires both MAMPs signals and wound signals. The transcriptomic data in this study indicates that numerous MAMP- and wounding-responsive genes were up-regulated by the exposure of E . aerogenes VCs. Besides, expression of immunity-related genes have similar pattern in response to E . aerogenes VCs and flg22. Collectively, the data in this study suggests that plant-deleterious mVCs activate plant immune responses via MAMPs and wound signals. Phytocytokines are the signal factors that are generated in response to wounding and pathogen infections (Tanaka & Heil 2021). The data in this study indicates that 11 SCOOPs, 2 PEPs and 2 PIPs were up-regulated after E . aerogenes VCs exposure ( Table S3 ). The expression of PEPR1 and PEPR2 has been found to be transcriptionally up-regulated by wounding (Yamaguchi et al . 2010). Pep2 is the phytocytokine which is perceived by both PEPR1 and PEPR2 and activates the defense responses (Yamaguchi et al . 2010). The data in this study indicates that the transcript abundance of pep2 could also be transactivated by E . aerogenes VCs. The transactivation of wound inducible PRRs (EFR, RLP23, LORE) might also suggest the involvement of the wounding response in the E . aerogenes VCs induced responses. The result suggestes that MAMPs and the wounding response might contribute to the induction of E . aerogenes VCs activated plant immunity ( Figure 7 ). STZ is the regulator of abiotic stress tolerance (Sakamoto et al . 2004; Mittler et al . 2006). In this study, STZ was identified as the hub genes within E . aerogenes VCs up-regulated genes. Other hub genes including MPK3, MPK11, WRKY40, WRKY33, XLG2, SOBIR1, SYP122, CNI1, AT1G19020 are the important components in plant immune signaling and disease resistance (Zhang et al . 2007b; Zhu et al . 2009; Pandey, Roccaro, Schön, Logemann & Somssich 2010; Bethke et al . 2012; Birkenbihl, Diezel & Somssich 2012; Maekawa et al . 2012; Takahashi, Shibuya & Ishikawa 2016; Lang, Genot, Hirt & Colcombet 2017; Dutta et al . 2020). The result suggests that STZ might also contribute to the regulation of plant immunity. Plant systemic immunity triggered by pathogens is defined as SAR, while the beneficial microbes activated immunity is defined as ISR (Pieterse et al . 2014). The activation of Pathogen-related gene 1 (PR1) is commonly characterized as SAR activation (Ryals et al. 1996; Loon, Rep & Pieterse 2006). Here, the data in this study shows that PR1 was not transactivated by E . aerogenes VCs exposure ( Table S1 ). Pseudomonas simiae WCS417 induced ISR is one of the well-known cases for the beneficial microbes activated ISR (Pieterse et al . 2021). The comparison of E . aerogenes VCs- and WCS417 related elicitors-transactivated genes (Stringlis et al . 2018a) shows that 74.8 % of E . aerogenes VCs inducible genes also respond to WCS417 related elicitors ( Figure S3 ). Besides, the data in this study also shows that MBY72 and FRO3 were up-regulated by E . aerogenes VCs. MYB72 is the regulator of ISR induction (Van der Ent et al . 2008). The gene expression of FRO3 would be up-regulated in an iron deficiency response (Mukherjee, Campbell, Ash & Connolly 2006). The result suggests that an ISR-like response was induced in MYB72 dependent manner by E . aerogenes VCs ( Figure 7 ). To prove E . aerogenes VCs-induced ISR-like response, the disease resistance in Arabidopsis seedlings with and without the pretreatment of E . aerogenes VCs were analyzed. The data shows that Arabidopsis seedlings with the E . aerogenes VCs treatment are more resistant against the pathogen infection ( Figure S4 ). The data from Figure 5c indicates that MKK3 is the regulator of the E . aerogenes VCs-induced MYB72 transactivation. The result suggests that MKK3 is the central components in the activation of E . aerogenes VCs-induced ISR-like response ( Figure 7 ). 4.4 E . aerogenes VCs promote the induction of plant cry-for-help strategy In response to the environmental stresses, plants have evolved a “cry-for-help” strategy which refers to the phenomenon that plants would release secondary metabolites or volatiles to recruit beneficial microbes to be equipped for a better adaptation, for protection against environmental stresses, or for nutrient intake (Pieterse et al. 2014; Jacoby, Peukert, Succurro, Koprivova & Kopriva 2017; Cheng, Zhang & He 2019; Rizaludin, Stopnisek, Raaijmakers & Garbeva 2021). Furthermore, it has been mentioned that the secretion of camalexin and coumarin could lead to the reshaping of plant microbiota (Jacoby, Koprivova & Kopriva 2021). CYP71A13 and PAD3 are the enzymes involved in camalexin biosynthesis (Zhou, Tootle & Glazebrook 1999; Schuhegger et al . 2006; Nafisi et al . 2007). The RNA-seq data from this study demonstrates that the transcript abundance of CYP71A13 and PAD3 was enhanced after E . aerogenes VCs exposure. MYB72 which modulates the activation of ISR is also the regulator of coumarin secretion (Van der Ent et al . 2008; Stringlis et al . 2018b). F6’H1 and BGLU42 are the enzymes which catalyze the biosynthesis and excretion of coumarin scopoletin (Clemens & Weber 2016; Stringlis et al . 2018b). The RNA-seq data from this study demonstrates that the transcript abundance of MYB72 and F6’H1 was enhanced after the E . aerogenes VCs exposure. The up-regulation of camalexin and coumarin biosynthesis genes could lead to the enhancement of the activity of camalexin and coumarin biosynthesis which suggest the increase of plant antimicrobial activity and the recruitment of beneficial microbes. Collectivelly, the data suggests that E . aerogenes VCs could function as the inducer of plant disease resistance enhancement and initiate the reassembly of plant microbiota. In addition, growing evidence has uncovered that the ISR inducing microbes also lead to the secretion of antimicrobial phytoalexin camalexin and coumarin (Contreras-Cornejo, Macı́as-Rodrı́guez, Beltrán-Peña, Herrera-Estrella & López-Bucio 2011; Stringlis et al. 2018b). The data in Stringlis et al . (2018b) indicate that the ISR-inducing beneficial microbes would cause the secretion of coumarin and influence the plant microbiota composition. In this study, the results suggest that plant-deleterious mVCs could also induce the ISR-like response in Arabidopsis . Furthermore, the MYB72-dependent ISR-like response might promote the activation of cry-for-help strategy in Arabidopsis . 4.5 MKK1/3 cascades contribute to the transactivation of E . aerogenes VCs-induced phytoalexin biosynthesis genes and the induction of callose deposition Evidence from the previous study revealed that cell wall damage is regulated by cell wall integrity response to wounding and leads to the activation of MTI signaling components (Engelsdorf et al . 2018). It has been demonstrated that MPK1-mediated cell wall integrity contributes to the cell protection in E . aerogenes VCs-induced cytotoxicity in yeast (Wu et al . 2019). The comparison of E . aerogenes VCs inducible genes and wound inducible genes indicated that 53 % of E . aerogenes VCs inducible genes after long mVCs exposure were also wound inducible genes. MKK1 and MKK3 are the signaling components activated by wounding (Morris, Guerrier, Leung & Giraudat 1997; Hadiarto et al. 2006; Takahashi et al. 2007). Antimicrobial substrates including antimicrobial peptides and phytoalexins serve as an important frontline defense of apoplastic space in plants. Previous studies reported that MKK1 and MKK3 are involved in pathogen-induced antimicrobial peptide PDF1.2 induction (Penninckx et al . 1998; Takahashi et al . 2007; Qiu et al . 2008). In this study, the data demonstrates that E . aerogenes VCs could also induce PDF1.2 via the regulation of MKK1 and MKK3. Moreover, the data from this study shows that the transcript abundance of camalexin biosynthesis genes, CYP71A13, and PAD3 were less up-regulated after E . aerogenes VCs exposure in mkk1 , mkk3 mutants. The transcript abundance of coumarin biosynthesis genes, MYB72, and F6’H1 were less up-regulated after E . aerogenes VCs exposure in the mkk3 mutant. The results suggest that MKK1 and MKK3 might be the important modulators for the biosynthesis of the antimicrobial substrates when exposed to microbial volatiles. However, MKK1 and MKK3 might play different roles in the transactivation of camalexin and coumarin biosynthesis genes ( Figure 7 ). MKK3 is the central components in the activation of E . aerogenes VCs-induced ISR-like response which induce the secretion of phytoalexin ( Figure 7 ). Due to the evidence that camalexin and coumarin could result in the plant microbiota reassembly (Jacoby et al . 2021). The results from this data might suggest that MKK1 and MKK3 are regulators which drive the plant’s cry for help strategy. Collectively, the data in this study shows that hypoxia response is the early E . aerogenes VCs induced response which might mount the later defense response in Arabidopsis . Moreover, E . aerogenes VCs act as the mediator to enhance both plant tissue and molecular defense and activate the cry for help mechanisms via the regulation of MAMP signaling and the wounding response ( Figure 7 ). More importantly, MKK3 positively regulates the E . aerogenes VCs induced ISR-like response, tissue defense and molecular defense; while MKK1 regulates the tissue defense and molecular defense in response to E . aerogenes VCs treatment ( Figure 7 ). It will be interesting to further investigate the specific roles of plant-deleterious mVCs-induced PRRs in defense response of plants to deleterious mVCs. Acknowledgements This work was supported by the Ministry of Science and Technology of Taiwan (MOST-106-2311-B-006-006-MY3, which organized by Dr. Hao-Jen Huang), Higher Education Sprout Project from Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University. Dr. Masaru Ohme-Takagi was supported by Yushan Fellow Program from Ministry of Education. Dr. Tetsuro Mimura was supported by NCKU 90 and Beyond Recruiting project from National Cheng Kung University. Conflict of interest The authors declare no potential conflict of interest. References Ahuja I., Kissen R. & Bones A.M. (2012). Phytoalexins in defense against pathogens. Trends in plant science , 17, 73–90. Albert I., Hua C., Nürnberger T., Pruitt R.N. & Zhang L. (2020). Surface sensor systems in plant immunity. Plant physiology , 182, 1582–1596. Armstrong W., Webb T., Darwent M. & Beckett P.M. (2009). Measuring and interpreting respiratory critical oxygen pressures in roots. Annals of Botany , 103, 281–293. Asai T., Tena G., Plotnikova J., Willmann M.R., Chiu W.-L., Gomez-Gomez L., … Sheen J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature , 415, 977–983. Bacete L., Mélida H., Miedes E. & Molina A. (2018). Plant cell wall-mediated immunity: Cell wall changes trigger disease resistance responses. The Plant Journal , 93, 614–636. Bailey-Serres J. & Voesenek L. (2008). Flooding stress: Acclimations and genetic diversity. Annu. Rev. Plant Biol ., 59, 313–339. Bari R. & Jones J.D. (2009). Role of plant hormones in plant defence responses. Plant molecular biology , 69, 473–488. Barnawal D., Bharti N., Pandey S.S., Pandey A., Chanotiya C.S. & Kalra A. (2017). Plant growth-promoting rhizobacteria enhance wheat salt and drought stress tolerance by altering endogenous phytohormone levels and TaCTR1/TaDREB2 expression. Physiologia plantarum , 161, 502–514. Baxter A., Mittler R. & Suzuki N. (2014). ROS as key players in plant stress signalling. Journal of experimental botany , 65, 1229–1240. Bethke G., Pecher P., Eschen-Lippold L., Tsuda K., Katagiri F., Glazebrook J., … Lee J. (2012). Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22. Molecular Plant-Microbe Interactions , 25, 471–480. Birkenbihl R.P., Diezel C. & Somssich I.E. (2012). Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection. Plant physiology , 159, 266–285. Bolger A.M., Lohse M. & Usadel B. (2014). Trimmomatic: A flexible trimmer for illumina sequence data. Bioinformatics , 30, 2114–2120. Bowen G. & Rovira A. (1999). The rhizosphere and its management to improve plant growth. Advances in agronomy , 66, 1–102. Campos M.L., Souza C.M. de, Oliveira K.B.S. de, Dias S.C. & Franco O.L. (2018). The role of antimicrobial peptides in plant immunity. Journal of experimental botany , 69, 4997–5011. Cheng Y.T., Zhang L. & He S.Y. (2019). Plant-microbe interactions facing environmental challenge. Cell host & microbe , 26, 183–192. Clemens S. & Weber M. (2016). The essential role of coumarin secretion for Fe acquisition from alkaline soil. Plant Signaling & Behavior , 11, e1114197. Contreras-Cornejo H.A., Macı́as-Rodrı́guez L., Beltrán-Peña E., Herrera-Estrella A. & López-Bucio J. (2011). Trichoderma -induced plant immunity likely involves both hormonal-and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance against necrotrophic fungi Botrytis cinerea . Plant signaling & behavior , 6, 1554–1563. Cordovez V., Mommer L., Moisan K., Lucas-Barbosa D., Pierik R., Mumm R., … Raaijmakers J.M. (2017). Plant phenotypic and transcriptional changes induced by volatiles from the fungal root pathogen Rhizoctonia solani . Frontiers in plant science , 8, 1262. Cordovez V., Schop S., Hordijk K., Dupré de Boulois H., Coppens F., Hanssen I., … Carrión V.J. (2018). Priming of plant growth promotion by volatiles of root-associated Microbacterium spp. Applied and environmental microbiology , 84, e01865–18. D’Alessandro M., Erb M., Ton J., Brandenburg A., Karlen D., Zopfi J. & Turlings T.C. (2014). Volatiles produced by soil-borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions. Plant, cell & environment , 37, 813–826. Dongen J.T. van, Fröhlich A., Ramı́rez-Aguilar S.J., Schauer N., Fernie A.R., Erban A., … Geigenberger P. (2009). Transcript and metabolite profiling of the adaptive response to mild decreases in oxygen concentration in the roots of Arabidopsis plants. Annals of botany , 103, 269–280. Dóczi R., Brader G., Pettkó-Szandtner A., Rajh I., Djamei A., Pitzschke A., … Hirt H. (2007) The arabidopsis mitogen-activated protein kinase kinase mkk3 is upstream of group c mitogen-activated protein kinases and participates in pathogen signaling. The Plant Cell 19, 3266–3279. Durrant W.E. & Dong X. (2004). Systemic acquired resistance. Annu. Rev. Phytopathol , 42, 185–209. Dutta A., Choudhary P., Gupta-Bouder P., Chatterjee S., Liu P.-P., Klessig D.F. & Raina R. (2020). Arabidopsis small defense-associated protein 1 modulates pathogen defense and tolerance to oxidative stress. Frontiers in Plant Science , 703. Ebel J. (1986). Phytoalexin synthesis: The biochemical analysis of the induction process. Annual review of phytopathology , 24, 235–264. Engelsdorf T., Gigli-Bisceglia N., Veerabagu M., McKenna J.F., Vaahtera L., Augstein F., … Hamann T. (2018). The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana . Science Signaling , 11, eaao3070. Esparza-Reynoso S., Ruı́z-Herrera L.F., Macı́as-Rodrı́guez L.I., Martı́nez-Trujillo M., López-Coria M., Sánchez-Nieto S., … López-Bucio J. (2021). Trichoderma atroviride -emitted volatiles improve growth of Arabidopsis seedlings through modulation of sucrose transport and metabolism. Plant, Cell & Environment . Finkel O.M., Salas-González I., Castrillo G., Conway J.M., Law T.F., Teixeira P.J.P.L., … Dangl J.L. (2020). A single bacterial genus maintains root growth in a complex microbiome. Nature , 587, 103–108. Franceschini A., Szklarczyk D., Frankild S., Kuhn M., Simonovic M., Roth A., … Jensen L.J. (2012). STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Research , 41, D808–D815. Garcı́a-Gómez P., Bahaji A., Gámez-Arcas S., Muñoz F.J., Sánchez-López Á.M., Almagro G., … others (2020). Volatiles from the fungal phytopathogen Penicillium aurantiogriseum modulate root metabolism and architecture through proteome resetting. Plant, Cell & Environment , 43, 2551–2570. Godson A. & Hoorn R.A. van der (2021). The front line of defence: A meta-analysis of apoplastic proteases in plant immunity. Journal of Experimental Botany , 72, 3381–3394. Gómez-Gómez L. & Boller T. (2000). FLS2: An LRR receptor–like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis . Molecular cell , 5, 1003–1011. Gururani M.A., Upadhyaya C.P., Baskar V., Venkatesh J., Nookaraju A. & Park S.W. (2013). Plant growth-promoting rhizobacteria enhance abiotic stress tolerance in solanum tuberosum through inducing changes in the expression of ROS-scavenging enzymes and improved photosynthetic performance. Journal of plant growth regulation , 32, 245–258. Hadiarto T., Nanmori T., Matsuoka D., Iwasaki T., Sato K.-i., Fukami Y., … Yasuda T. (2006). Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1–AtMEK1 pathway by wounding. Planta , 223, 708–713. Han S.H., Lee S.J., Moon J.H., Park K.H., Yang K.Y., Cho B.H., … others (2006). GacS-dependent production of 2R, 3R-butanediol by Pseudomonas chlororaphis O6 is a major determinant for eliciting systemic resistance against Erwinia carotovora but not against Pseudomonas syringae pv. tabaci in tobacco. Molecular Plant-Microbe Interactions , 19, 924–930. Hander T., Fernández-Fernández Á.D., Kumpf R.P., Willems P., Schatowitz H., Rombaut D., … others (2019). Damage on plants activates CA 2+ -dependent metacaspases for release of immunomodulatory peptides. Science , 363. Hann C.T., Bequette C.J., Dombrowski J.E. & Stratmann J.W. (2014). Methanol and ethanol modulate responses to danger-and microbe-associated molecular patterns. Frontiers in Plant Science , 5, 550. Hao H.-T., Zhao X., Shang Q.-H., Wang Y., Guo Z.-H., Zhang Y.-B., … Wang R.-Y. (2016). Comparative digital gene expression analysis of the Arabidopsis response to volatiles emitted by Bacillus amyloliquefaciens . PLoS One , 11, e0158621. Hilu K.W. & Randall J.L. (1984). Convenient method for studying grass leaf epidermis. Taxon , 33, 413–415. Hou S., Liu D. & He P. (2021). Phytocytokines function as immunological modulators of plant immunity. Stress Biology , 1, 1–14. Hsu F.-C., Chou M.-Y., Chou S.-J., Li Y.-R., Peng H.-P. & Shih M.-C. (2013). Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis . The Plant Cell , 25, 2699–2713. Huang C.-J., Tsay J.-F., Chang S.-Y., Yang H.-P., Wu W.-S. & Chen C.-Y. (2012). Dimethyl disulfide is an induced systemic resistance elicitor produced by Bacillus cereus c1l. Pest management science , 68, 1306–1310. Ikeuchi M., Iwase A., Rymen B., Lambolez A., Kojima M., Takebayashi Y., … others (2017). Wounding triggers callus formation via dynamic hormonal and transcriptional changes. Plant physiology , 175, 1158–1174. Jacoby R., Peukert M., Succurro A., Koprivova A. & Kopriva S. (2017). The role of soil microorganisms in plant mineral nutrition—current knowledge and future directions. Frontiers in plant science , 8, 1617. Jacoby R.P., Koprivova A. & Kopriva S. (2021). Pinpointing secondary metabolites that shape the composition and function of the plant microbiome. Journal of Experimental Botany , 72, 57–69. Jalali F., Zafari D. & Salari H. (2017). Volatile organic compounds of some Trichoderma spp. Increase growth and induce salt tolerance in Arabidopsis thaliana . Fungal ecology , 29, 67–75. Kanchiswamy C.N., Malnoy M. & Maffei M.E. (2015). Chemical diversity of microbial volatiles and their potential for plant growth and productivity. Frontiers in plant science , 6, 151. Kim N.Y., Jang Y.J. & Park O.K. (2018). AP2/ERF family transcription factors ORA59 and RAP2.3 interact in the nucleus and function together in ethylene responses. Frontiers in plant science , 9, 1675. Kishimoto K., Matsui K., Ozawa R. & Takabayashi J. (2005). Volatile C6-aldehydes and Allo-ocimene activate defense genes and induce resistance against Botrytis cinerea in Arabidopsis thaliana . Plant and Cell Physiology , 46, 1093–1102. Lang J., Genot B., Hirt H. & Colcombet J. (2017). Constitutive activity of the Arabidopsis MAP Kinase 3 confers resistance to Pseudomonas syringae and drives robust immune responses. Plant Signaling & Behavior , 12, e1356533. Lee B., Farag M.A., Park H.B., Kloepper J.W., Lee S.H. & Ryu C.-M. (2012). Induced resistance by a long-chain bacterial volatile: Elicitation of plant systemic defense by a C13 volatile produced by Paenibacillus polymyxa . PloS one , 7, e48744. Li J., Ezquer I., Bahaji A., Montero M., Ovecka M., Baroja-Fernández E., … others (2011). Microbial volatile-induced accumulation of exceptionally high levels of starch in Arabidopsis leaves is a process involving NTRC and starch synthase classes iii and iv. Molecular Plant-Microbe Interactions , 24, 1165–1178. Li N., Wang W., Bitas V., Subbarao K., Liu X. & Kang S. (2018). Volatile compounds emitted by diverse verticillium species enhance plant growth by manipulating auxin signaling. Molecular Plant-Microbe Interactions , 31, 1021–1031. Li Q., Wang C. & Mou Z. (2020). Perception of damaged self in plants. Plant physiology , 182, 1545–1565. Loon L.C. van, Rep M. & Pieterse C.M. (2006). Significance of inducible defense-related proteins in infected plants. Annu. Rev. Phytopathol , 44, 135–162. Maekawa S., Sato T., Asada Y., Yasuda S., Yoshida M., Chiba Y. & Yamaguchi J. (2012). The Arabidopsis ubiquitin ligases ATL31 and ATL6 control the defense response as well as the carbon/nitrogen response. Plant molecular biology , 79, 217–227. Melotto M., Underwood W., Koczan J., Nomura K. & He S.Y. (2006). Plant stomata function in innate immunity against bacterial invasion. Cell , 126, 969–980. Millet Y.A., Danna C.H., Clay N.K., Songnuan W., Simon M.D., Werck-Reichhart D. & Ausubel F.M. (2010). Innate immune responses activated in Arabidopsis roots by microbe-associated molecular patterns. The Plant Cell , 22, 973–990. Mine A., Seyfferth C., Kracher B., Berens M.L., Becker D. & Tsuda K. (2018). The defense phytohormone signaling network enables rapid, high-amplitude transcriptional reprogramming during effector-triggered immunity. The Plant Cell , 30, 1199–1219. Mittler R., Kim Y., Song L., Coutu J., Coutu A., Ciftci-Yilmaz S., … Zhu J.-K. (2006). Gain-and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS letters , 580, 6537–6542. Miya A., Albert P., Shinya T., Desaki Y., Ichimura K., Shirasu K., … Shibuya N. (2007). CERK1 , a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis . Proceedings of the National Academy of Sciences , 104, 19613–19618. Morcillo R.J., Singh S.K., He D., An G., Vı́lchez J.I., Tang K., … others (2020). Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner. The EMBO journal , 39, e102602. Morris P.C., Guerrier D., Leung J. & Giraudat J. (1997). Cloning and characterisation of MEK1, an Arabidopsis gene encoding a homologue of MAP kinase kinase. Plant molecular biology , 35, 1057–1064. Mukherjee I., Campbell N.H., Ash J.S. & Connolly E.L. (2006). Expression profiling of the Arabidopsis ferric chelate reductase ( FRO ) gene family reveals differential regulation by iron and copper. Planta , 223, 1178–1190. Mustroph A., Lee S.C., Oosumi T., Zanetti M.E., Yang H., Ma K., … Bailey-Serres J. (2010). Cross-kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses. Plant Physiology , 152, 1484–1500. Nafisi M., Goregaoker S., Botanga C.J., Glawischnig E., Olsen C.E., Halkier B.A. & Glazebrook J. (2007). Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis. The Plant Cell , 19, 2039–2052. Nguyen H.M., Sako K., Matsui A., Suzuki Y., Mostofa M.G., Ha C.V., … Seki M. (2017). Ethanol enhances high-salinity stress tolerance by detoxifying reactive oxygen species in Arabidopsis thaliana and rice. Frontiers in Plant Science , 8, 1001. Nieto-Jacobo M.F., Steyaert J.M., Salazar-Badillo F.B., Nguyen D.V., Rostás M., Braithwaite M., … others (2017). Environmental growth conditions of Trichoderma spp. Affects indole acetic acid derivatives, volatile organic compounds, and plant growth promotion. Frontiers in plant science , 8, 102. Pandey S.P., Roccaro M., Schön M., Logemann E. & Somssich I.E. (2010). Transcriptional reprogramming regulated by wrky18 and WRKY40 facilitates powdery mildew infection of Arabidopsis . The Plant Journal , 64, 912–923. Park Y.-S., Dutta S., Ann M., Raaijmakers J.M. & Park K. (2015). Promotion of plant growth by Pseudomonas fluorescens strain ss101 via novel volatile organic compounds. Biochemical and Biophysical Research Communications , 461, 361–365. Peng Y., Wersch R. van & Zhang Y. (2018). Convergent and divergent signaling in PAMP-triggered immunity and effector-triggered immunity. Molecular plant-microbe interactions , 31, 403–409. Penninckx I.A., Thomma B.P., Buchala A., Métraux J.-P. & Broekaert W.F. (1998). Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis . The Plant Cell , 10, 2103–2113. Pieterse C.M., Berendsen R.L., Jonge R. de, Stringlis I.A., Van Dijken A.J., Van Pelt J.A., … Bakker P.A. (2021). Pseudomonas simiae wcs417: Star track of a model beneficial rhizobacterium. Plant and Soil , 461, 245–263. Pieterse C.M., Zamioudis C., Berendsen R.L., Weller D.M., Van Wees S.C. & Bakker P.A. (2014). Induced systemic resistance by beneficial microbes. Annual review of phytopathology , 52, 347–375. Pieterse C., Van Wees S., Ton J., Van Pelt J. & Van Loon L. (2002). Signalling in rhizobacteria-induced systemic resistance in Arabidopsis thaliana . Plant biology , 4, 535–544. Pii Y., Mimmo T., Tomasi N., Terzano R., Cesco S. & Crecchio C. (2015). Microbial interactions in the rhizosphere: Beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. A review. Biology and fertility of soils , 51, 403–415. Porter K., Shimono M., Tian M. & Day B. (2012). Arabidopsis actin-depolymerizing factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics. PLoS Pathog , 8, e1003006. Qi J., Wang J., Gong Z. & Zhou J.-M. (2017). Apoplastic ROS signaling in plant immunity. Current opinion in plant biology , 38, 92–100. Qiu J.-L., Zhou L., Yun B.-W., Nielsen H.B., Fiil B.K., Petersen K., … Morris P.C. (2008). Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant physiology , 148, 212–222. Rizaludin M.S., Stopnisek N., Raaijmakers J.M. & Garbeva P. (2021). The chemistry of stress: Understanding the ‘cry for help’ of plant roots. Metabolites , 11, 357. Ryals J.A., Neuenschwander U.H., Willits M.G., Molina A., Steiner H.-Y. & Hunt M.D. (1996). Systemic acquired resistance. The plant cell , 8, 1809. Ryu C.-M., Farag M.A., Hu C.-H., Reddy M.S., Kloepper J.W. & Paré P.W. (2004). Bacterial volatiles induce systemic resistance in Arabidopsis . Plant physiology , 134, 1017–1026. Ryu C.-M., Farag M.A., Hu C.-H., Reddy M.S., Wei H.-X., Paré P.W. & Kloepper J.W. (2003). Bacterial volatiles promote growth in Arabidopsis . Proceedings of the National Academy of Sciences , 100, 4927–4932. Sakamoto H., Maruyama K., Sakuma Y., Meshi T., Iwabuchi M., Shinozaki K. & Yamaguchi-Shinozaki K. (2004). Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant physiology , 136, 2734–2746. Sako K., Sunaoshi Y., Tanaka M., Matsui A. & Seki M. (2018). The duration of ethanol-induced high-salinity stress tolerance in Arabidopsis thaliana . Plant signaling & behavior , 13, e1500065. Schmittgen T.D. & Livak K.J. (2008). Analyzing real-time PCR data by the comparative ct method. Nature protocols , 3, 1101. Schuhegger R., Nafisi M., Mansourova M., Petersen B.L., Olsen C.E., Svatos A., … Glawischnig E. (2006). CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis. Plant physiology , 141, 1248–1254. Song G.C., Jeon J.-S., Sim H.-J., Lee S., Jung J., Kim S.-G., … Ryu C.-M. (2022). Dual functionality of natural mixtures of bacterial volatile compounds on plant growth. Journal of experimental botany , 73, 571–583. Souza R. de, Ambrosini A. & Passaglia L.M. (2015). Plant growth-promoting bacteria as inoculants in agricultural soils. Genetics and molecular biology , 38, 401–419. Stringlis I.A., Proietti S., Hickman R., Van Verk M.C., Zamioudis C. & Pieterse C.M. (2018a). Root transcriptional dynamics induced by beneficial rhizobacteria and microbial immune elicitors reveal signatures of adaptation to mutualists. The Plant Journal , 93, 166–180. Stringlis I.A., Yu K., Feussner K., De Jonge R., Van Bentum S., Van Verk M.C., … Pieterse C.M. (2018b). MYB72-dependent coumarin exudation shapes root microbiome assembly to promote plant health. Proceedings of the National Academy of Sciences , 115, E5213–E5222. Sukkasem P., Kurniawan A., Kao T.-C. & Chuang H.-w. (2018). A multifaceted rhizobacterium bacillus licheniformis functions as a fungal antagonist and a promoter of plant growth and abiotic stress tolerance. Environmental and experimental botany , 155, 541–551. Sun L., Cao M., Liu F., Wang Y., Wan J., Wang R., … Xu J. (2020). The volatile organic compounds of Floccularia luteovirens modulate plant growth and metabolism in Arabidopsis thaliana . Plant and Soil , 456, 207–221. Takahashi F., Yoshida R., Ichimura K., Mizoguchi T., Seo S., Yonezawa M., … Shinozaki K. (2007). The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis . The Plant Cell , 19, 805–818. Takahashi T., Shibuya H. & Ishikawa A. (2016). SOBIR1 contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis . Bioscience, biotechnology, and biochemistry , 80, 1577–1579. Tanaka K. & Heil M. (2021). Damage-associated molecular patterns (DAMPs) in plant innate immunity: Applying the danger model and evolutionary perspectives. Annual Review of Phytopathology , 59. Teixeira P.J., Colaianni N.R., Law T.F., Conway J.M., Gilbert S., Li H., … others (2021). Specific modulation of the root immune system by a community of commensal bacteria. Proceedings of the National Academy of Sciences , 118. Trapnell C., Roberts A., Goff L., Pertea G., Kim D., Kelley D.R., … Pachter L. (2012). Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature protocols , 7, 562–578. Valeri M.C., Novi G., Weits D.A., Mensuali A., Perata P. & Loreti E. (2021). Botrytis cinerea induces local hypoxia in Arabidopsis leaves. New Phytologist , 229, 173–185. Van der Ent S., Verhagen B.W., Van Doorn R., Bakker D., Verlaan M.G., Pel M.J., … others (2008). MYB72 is required in early signaling steps of rhizobacteria-induced systemic resistance in Arabidopsis . Plant Physiology , 146, 1293–1304. Van Loon L., Bakker P. & Pieterse C. (1998). Systemic resistance induced by rhizosphere bacteria. Annual review of phytopathology , 36, 453–483. Vicente J., Mendiondo G.M., Pauwels J., Pastor V., Izquierdo Y., Naumann C., … others (2019). Distinct branches of the N-end rule pathway modulate the plant immune response. New Phytologist , 221, 988–1000. Vurukonda S.S.K.P., Vardharajula S., Shrivastava M. & SkZ A. (2016). Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria. Microbiological research , 184, 13–24. Weisskopf L., Schulz S. & Garbeva P. (2021). Microbial volatile organic compounds in intra-kingdom and inter-kingdom interactions. Nature Reviews Microbiology , 19, 391–404. Wenke K., Kopka J., Schwachtje J., Van Dongen J. & Piechulla B. (2019). Volatiles of rhizobacteria Serratia and Stenotrophomonas alter growth and metabolite composition of Arabidopsis thaliana . Plant Biology , 21, 109–119. Wenke K., Wanke D., Kilian J., Berendzen K., Harter K. & Piechulla B. (2012). Volatiles of two growth-inhibiting rhizobacteria commonly engage AtWRKY18 function. The Plant Journal , 70, 445–459. Wu P.-H., Ho Y.-L., Ho T.-S., Chang C.-H., Ye J.-C., Wang C.-H., … Liu C.-C. (2019). Microbial volatile compounds-induced cytotoxicity in the yeast Saccharomyces cerevisiae : The role of MAPK signaling and proteasome regulatory pathway. Chemosphere , 233, 786–795. Yamaguchi Y., Huffaker A., Bryan A.C., Tax F.E. & Ryan C.A. (2010). PEPR2 is a second receptor for the pep1 and pep2 peptides and contributes to defense responses in Arabidopsis. The Plant Cell , 22, 508–522. Yasuda M., Ishikawa A., Jikumaru Y., Seki M., Umezawa T., Asami T., … others (2008). Antagonistic interaction between systemic acquired resistance and the abscisic acid–mediated abiotic stress response in Arabidopsis . The Plant Cell , 20, 1678–1692. Yi S.Y., Shirasu K., Moon J.S., Lee S.-G. & Kwon S.-Y. (2014). The activated SA and JA signaling pathways have an influence on flg22-triggered oxidative burst and callose deposition. PloS one , 9, e88951. Yuan M., Ngou B.P.M., Ding P. & Xin X.-F. (2021). PTI-ETI crosstalk: An integrative view of plant immunity. Current Opinion in Plant Biology , 62, 102030. Zamioudis C., Hanson J. & Pieterse C.M. (2014). β-Glucosidase BGLU42 is a MYB72-dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots. New Phytologist , 204, 368–379. Zhang H., Kim M.-S., Krishnamachari V., Payton P., Sun Y., Grimson M., … others (2007a). Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis . Planta , 226, 839–851. Zhang H., Xie X., Kim M.-S., Kornyeyev D.A., Holaday S. & Paré P.W. (2008). Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisic acid levels in planta. The Plant Journal , 56, 264–273. Zhang Z., Feechan A., Pedersen C., Newman M.-A., Qiu J.-l., Olesen K.L. & Thordal-Christensen H. (2007b). A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways. The Plant Journal , 49, 302–312. Zhao Y., Wei T., Yin K.-Q., Chen Z., Gu H., Qu L.-J. & Qin G. (2012). Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses. New Phytologist , 195, 450–460. Zhou F., Emonet A., Tendon V.D., Marhavy P., Wu D., Lahaye T. & Geldner N. (2020). Co-incidence of damage and microbial patterns controls localized immune responses in roots. Cell , 180, 440–453. Zhou J.-M. & Zhang Y. (2020). Plant immunity: Danger perception and signaling. Cell , 181, 978–989. Zhou J., Sun A. & Xing D. (2013). Modulation of cellular redox status by thiamine-activated NADPH oxidase confers Arabidopsis resistance to Sclerotinia sclerotiorum . Journal of experimental botany , 64, 3261–3272. Zhou N., Tootle T.L. & Glazebrook J. (1999). Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monooxygenase. The Plant Cell , 11, 2419–2428. Zhu H., Li G.-J., Ding L., Cui X., Berg H., Assmann S.M. & Xia Y. (2009). Arabidopsis extra large G-protein 2 (XLG2) interacts with the gβ subunit of heterotrimeric G protein and functions in disease resistance. Molecular plant , 2, 513–525. Zipfel C., Kunze G., Chinchilla D., Caniard A., Jones J.D., Boller T. & Felix G. (2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts agrobacterium-mediated transformation. Cell , 125, 749–760. Figure 1 E. aerogenes VCs activate the tissue defense response in Arabidopsis thaliana . (a) Equipment of mVCs co-culture experiments. (b) Growth inhibition effect of Enterobacter aerogenes VCs on A. thaliana after 24 hr exposure. The results are representative of 3 biological replicates. Scale bar= 1 cm. (c) Measurement of Primary root length. Error bar shows the standard error. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (d) CM-H 2 DCFDA staining visualized ROS accumulation in the primary root of A . thaliana after exposure to E. aerogenes VCs for 1, 3 hr. The ROS contents were visualized by Leica DMLB fluorescence microscope equipped with a GFP filter. The results are representative of 3 biological replicates. Scale bar, 50 µm. (e) Callose deposition was induced by E. aerogenes VCs after 48 hr exposure. Callose deposition was detected by aniline blue staining and visualized by Leica DMLB fluorescent microscope equipped with a DAPI filter. The results are representative of 3 biological replicates. Scale bar, 100 µm. Figure 2 E. aerogenes VCs modulate root inhibition via auxin response in A . thaliana . (a) Expression of auxin responsive element DR5::GFP in Arabidopsis roots after 24 hr E . aerogenes co-culture. The results are representative of 3 biological replicates. Scale bar, 50 µm. (b) Expression and distribution of PINs in Arabidopsis roots after 24 hr E . aerogenes co-culture. Images of PIN3::PIN3-GFP , PIN4::PIN4-GFP expression in Arabidopsis root exposed to E . aerogenes VCs for 24 hr. The results are representative of 3 biological replicates. Scale bar, 50 µm.µm. Figure 3 E. aerogenes VCs induce rapid defense transcriptome. (a) The numbers of differentially expressed (DE) genes in A. thaliana in response to E. aerogenes VCs exposure for 3, 24 hr. The white bar indicates up-regulated genes, the grey bar indicates down-regulated genes. Genes with FPKM > 0, | log2(FC) | >= 1, p < 0.05 were identified as differentially expressed gene. (b-c) GO term enrichment analysis of mVCs up-regulated genes after E. aerogenes VCs exposure for 3 (B), 24 (C) hr. Red indicates higher statistical significance; yellow indicates lower statistical significance. The Bars refer to the percentage of the regulated gene numbers of each category in the total DE gene list. Black dots refer to the percentage of gene numbers of each category in the annotated genes in the genome. (d) Network component analysis for the hub proteins which are up-regulated by long E. aerogenes VCs exposure. The analysis was performed in STRING database with 24 hr E. aerogenes VCs up-regulated genes used as query. (e) Expression of innate immune genes, FRK1, WRKY33 after long E. aerogenes VCs exposure in A . thaliana . The mRNA abundances were examined by real-time reverse transcription-polymerase chain reaction (RT-qPCR). Actin 8 was used as the internal control. Data are the M±SE for 3 biological replicates for each treatment. Asterisks indicate the significant differences between control and mVCs exposure treatment. The student’s t-test was applied. *, P < 0.05; **, P < 0.01; ***, P < 0.001 Figure 4 E. aerogenes VCs inducible immune gene expression profiles are more similar to the flg22 inducible profiles. (a) Comparisons of the number of DE genes among E. aerogenes VCs inducible genes after long exposure, wounding- (Ikeuchi et al . 2017), and flg22-inducible (Stringlis et al . 2018a) genes. (b) The immune gene expression in response to E. aerogenes VCs, flg22 and wounding. Red indicates the up-regulated genes. Blue indicates the down-regulated genes. Black indicates the non-regulated genes. Figure 5 AtMKK1 and AtMKK3 are involved in the molecular defense genes transcription. (a) Transcript abundance of defensin gene PDF1.2 in mkk1 and mkk3 mutants after long E. aerogenes VCs exposure. (b) Transcript abundance of camalexin biosynthesis genes, CYP71A13 and PAD3, in mkk1 and mkk3 mutants after long E. aerogenes VCs exposure. (c) Transcript abundance of coumarin biosynthesis genes, MYB72 and F6’H1, in mkk1 and mkk3 mutants after long E. aerogenes VCs exposure. The mRNA abundances were examined by real-time reverse transcription-polymerase chain reaction (RT-qPCR). Actin 8 was used as the internal control. Data are the M±SE for 3 technical replicates for each treatment. All experiments were performed twice with similar results. Asterisks indicate the significant differences between control and VCs exposure treatment. The student’s t-test was applied. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 6 MKK1 and MKK3 mediate E. aerogenes VCs-induced callose deposition in roots. (a) The density of callose deposits after 48 hr E. aerogenes VCs exposure in mkk1 and mkk3 mutants. Callose deposition was detected by aniline blue staining and visualized by Leica DMLB fluorescent microscope equipped with a DAPI filter. The results are representative of 4 biological replicates. Scale bar, 100 µm. (b) The quantification of callose deposits in Arabidopsis roots after 48 hr E. aerogenes VCs exposure. Data are the M±SE for 3 biological replicates for each treatment. Asterisks indicate the significant differences between control and mVCs exposure treatment. The student’s t-test was applied. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 7 Proposed model of the MKK1- and MKK3-dependent regulatory mechanisms in response to E. aerogenes VCs in A . thaliana . Hypoxia response is induced rapidly after the deleterious E. aerogenes VCs treatment. After longer exposure, the wounding and MAMPs signalings are activated. MKK1 and MKK3 are involved in the regulation of the deleterious volatiles-induced callose deposition and camelexin biosynthesis. Moreover, MKK3 plays essential roles in the E. aerogenes VCs-induced coumarin biosynthesis and ISR activation. Table 1. E. aerogenes VCs inducible PRRs in A . thaliana . Table 2. The numbers of E. aerogenes VCs inducible antimicrobial substances. 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Keywords atmkk1 atmkk3 immune mvcs signaling volatile emissions Authors Affiliations Ching-Han Chang National Cheng Kung University View all articles by this author Wu-Guei Wang National Cheng Kung University View all articles by this author Pei-Yu Su National Cheng Kung University View all articles by this author Yu-Shuo Chen National Cheng Kung University View all articles by this author Tri-Phuong Nguyen National Cheng Kung University View all articles by this author Jian Xu Department of Plant Systems Physiology Radboud University Nijmegen The Netherlands View all articles by this author Masaru Ohme-Takagi National Cheng Kung University View all articles by this author Tetsuro Mimura National Cheng Kung University View all articles by this author Hao-Jen Huang 0000-0002-7459-162X [email protected] National Cheng Kung University View all articles by this author Metrics & Citations Metrics Article Usage 289 views 111 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Ching-Han Chang, Wu-Guei Wang, Pei-Yu Su, et al. 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