GhMAC3e is involved in plant growth and defense response to Verticillium dahliae

doi:10.21203/rs.3.rs-4863406/v1

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GhMAC3e is involved in plant growth and defense response to Verticillium dahliae

https://doi.org/10.21203/rs.3.rs-4863406/v1

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published 10 Oct, 2024

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The MOS4-Associated Complex (MAC) is a highly conserved protein complex involved in pre-mRNA splicing and spliceosome assembly, which plays a vital role in plant immunity. It comprises key components such as MOS4, CDC5, and PRL1. MAC3A/B, as U-box E3 ubiquitin ligases, are crucial for various plant processes including development, stress responses, and disease resistance. However, their roles in cotton remain largely unknown. In this study, we first cloned the GhMAC3e gene from cotton and explored its biological functions by using virus-induced gene silencing (VIGS) in cotton and transgenic overexpression in Arabidopsis. The results showed that GhMAC3e is ubiquitously expressed in cotton tissues and could be induced by salt stress, Verticillium dahliae (VD) infection, PEG, ABA, ETH, GA3, MeJA, and SA. Silencing GhMAC3e retarded primary stem growth and reduced biomass of cotton coupled with the reduced auxin content in the petioles and veins. Silencing GhMAC3e up-regulated expression of cell growth-related genes GhXTH16 and Gh3.6, while down-regulated GhSAUR12 expression. Ectopic expression of GhMAC3e in Arabidopsis significantly enhanced its resistance to Verticillium wilt (VW) in terms of decreased pathogen biomass and lowered plant mortality. Overexpression of GhMAC3e dramatically upregulated AtPR1 by around 15 fold and more than 262 fold under basal and VD inoculation condition, respectively. This change was not associated with the expression of GhNPR1. In conclusion, GhMAC3e may not only regulate plant growth by influencing auxin distribution and growth-related gene expression, but also play important roles in VW resistance via mediating SA signaling independent of NPR1 transcription level.

cotton

MOS4-Associated Complex

GhMAC3e

auxin

Verticillium dahliae

GhMAC3e expression was induced by various stresses and hormones. GhMAC3e may regulate plant growth by influencing auxin distribution, and play important roles in Verticillium wilt resistance via mediating SA signaling.

The MOS4-Associated Complex (MAC) has been previously identified as an evolutionarily conserved, multifunctional protein complex that plays a pivotal role in the activation of plant immunity (Jia, et al. 2023; Johnson, et al. 2011; Monaghan, et al. 2009; Palma, et al. 2007). MAC is the Arabidopsis orthologous complex of the NINETEEN COMPLEX (NTC) in yeast or PRE-mRNA PROCESSING FACTOR 19 complex (Prp19C) of human, and consists of more than 20 proteins. Many members of the MAC complex are involved in alternative splicing (AS) of mRNA(Mach 2017; Monaghan, et al. 2009; Zhang, et al. 2014). MOS4 is a fundamental member of the MOS4-associated complex, Cell Division Cycle5 (AtCDC5) is an atypical R2R3 MYB transcription factor, and Pleiotropic Regulatory Locus 1 (PRL1) is a conserved nuclear WD-40 protein. MOS4, CDC5, and PRL1 regulate plant development and immunity, and they are essential components involved in plant disease resistance signaling(Feng, et al. 2024; Lin, et al. 2007; Zhang, et al. 2014; Zhang, et al. 2013). MAC3A and MAC3B are two functionally redundant U-box E3 ubiquitin ligases homologous to PRP19 in Arabidopsis. As the core components of MAC, they are involved in splicing pre-mRNAs of R genes and thus required for plant immunity. Loss-of-function mutations of these genes exhibited higher susceptibility to virulent pathogens(Li, et al. 2019; Monaghan, et al. 2009).

MAC3A/B was reported to promote the biogenesis of miRNAs and siRNAs, which interact with the DCL1 complex to control miRNA levels through modulating pri-miRNA transcription, processing, and stability(Jia, et al. 2017; Li, et al. 2018). Previous genetic analyses reported that MAC3A/B plays an essential role in growth and development in Arabidopsis. MAC3A/B redundantly controls circadian period by facilitating the proper splicing of clock-related genes and regulate flowering time by substrate ubiquitylation(Feke, et al. 2019; Feke, et al. 2020). Meanwhile MAC3A/B ubiquitinate UBP14/DA3 to maintain ploidy level and organ size(Guo, et al. 2024), they also regulate seed longevity through alternative splicing(Niñoles, et al. 2022). MAC3A/3B interact with Cryptochromes to assemble into CRY condensates to positively regulate hypocotyl elongation in a blue light–dependent manner(Jiang, et al. 2023). MAC3A/B mediate ubiquitination and degradation of the transcription factor ERF13 to promote lateral root emergence(Yu, et al. 2024). However, little has been reported about the role of MAC3 in plants other than Arabidopsis.

Upland cotton (Gossypium hirsutum L.) produces staple renewable textile fibers, VW causes big loss of cotton yield and quality, breeding new cultivars resistant to this devasting disease with high yield potential is very important for cotton industry. In the present study, we cloned a cotton MAC3A/B homologous gene and named as GhMAC3e, the expression patterns of GhMAC3e in response to both stress hormones including ABA, ethylene (ETH), methyl jasmonate (MeJA), salicylic acid (SA), and the growth hormone of gibberellin (GA₃) were characterized. The biological function of this gene and the underlying molecular mechanisms were evaluated by using virus-induced gene silencing (VIGS) technology and transgenic overexpression of GhMAC3e in Arabidopsis.

MAC3 protein analogs in upland cotton genome

Using amino acid sequences of AtMAC3A and AtMAC3B as queries to search against cotton genome (https://www.cottongen.org/), six analogous proteins (Gh_A01G0193, Gh_D01G2267, Gh_A02G0417, Gh_D02G0470, Gh_A09G2001 and Gh_D09G2202) were found in upland cotton genome and named as GhMAC3a~GhMAC3f according to the chromosome on which each gene resides. Phylogenetic analysis revealed that cotton MAC3 homologous proteins belong to the same clade and are closely related to those of soybean and Arabidopsis, but relatively far away from MACs in rice, maize and wheat (Fig. 1A). Multiple sequence alignment analysis showed that GhMAC3e contain a conserved U-box domain, Prp19 domain, and WD40 domain, respectively, and the MAC3s of cotton, soybean and Arabidopsis exhibit a high degree of similarity, suggesting that the homologous proteins of MAC3 are evolutionarily conserved and possibly perform similar functions in dicotyledonous plants. GhMAC3e (Gh_A09G2001) consists of a 1, 575 bp open reading frame, encoding 524 amino acids.

This protein was originally identified in a fiberless cotton mutant by proteomics analysis, its differential accumulation in the wild type and fiberless mutant ovules suggested that this gene is relevant to fiber development(Liu, et al. 2012), and therefore was molecularly cloned for further function analysis.

Expression pattern of GhMAC3e

To evaluate the possible biological functions of MAC3 genes in cotton, we analyzed the spatiotemporal expression characteristics of GhMAC3s and changes in response to abiotic and biotic stresses, respectively, by using public RNA-seq database of G. hirsutum (Bio Project Accession: PRJNA490626). The results showed that GhMAC3a is predominantly expressed in stamen and the ovules at -3 days post-anthesis (DPA); GhMAC3d is highly expressed in stem; GhMAC3b, GhMAC3c, GhMAC3e, and GhMAC3f exhibited similar expression patterns between different tissues, with relatively higher levels in stem and the ovules before anthesis (Fig. 2A). Six GhMAC3s can be induced to express by salt (NaCl), PEG (Polyethylene Glycol), and VD strain Vd6, and roughly in a similar pattern (Fig. 2B). These results indicate that GhMAC3s may be involved in cotton growth, ovule development, and responses to different stresses.

As phytohormones play crucial roles in regulating plant growth and stress tolerance, we further analyzed if expression of GhMAC3e could be induced by exogenous GA₃ (representing growth hormone), and stress hormones by using RT-qPCR (Fig. 3). The results showed GhMAC3e could be significantly induced by all above phytohormone treatments: The expression of GhMAC3s was up-regulated by GA₃ after 3 hours post treatment (hpt) with a peak at 6 hpt (Fig. 3B); under ABA treatment, the abundance of GhMAC3e mRNAs at 1, 3, and 12 hpt dropped to a level that even lower than at 0 hpt, but peaked at 24 hpt to be 2.8 fold higher than that at 0 hpt (Fig. 3C); expression of GhMAC3e can be significantly induced by SA, MeJA, and ETH as early as 1 hpt, and up-regulated up to 27-, 23-, and 14- fold higher levels compared to that at 0 htp, respectively (Fig. 3D-F). These results indicate that GhMAC3e expression is swiftly response to both exogenous GA₃and stress hormones, suggesting that GhMAC3e may play regulatory roles both in growth and stress responses.

Silencing GhMAC3e compromised primary stem growth and biomass accumulation in cotton

To elucidate the biological function of GhMAC3e, we employed virus-induced gene silencing (VIGS) technology to knock down GhMAC3e in cotton. In this system, silencing GhCLA1 to produce an albino phenotype was set as a visible marker to monitor the efficiency of VIGS, cotton leaves began to display the expected albino phenotype at around 14 d post Agro-infiltration with the TRV1 and TRV2:GhCLA1 (Fig. S1A). So we harvested TRV2:00 (empty vector control, WT) and TRV2:GhMAC3e plants at 14 days post-infiltration to determine the expression level of GhMAC3e by semi-quantitative RT-PCR, respectively. The results showed that GhMAC3e mRNA level was significantly reduced in TRV2:GhMAC3 plants (Fig. S1B), indicating that GhMAC3e expression was effectively silenced. The plant heights of GhMAC3e-silenced plants were significantly reduced by 25.7% compared to the control plants (Fig.4A, B), notably, this significant reduction attributed to the main stem length loss (Fig.4C), because no significant difference was observed in hypocotyl length between GhMAC3e-silenced plants and the control plants (Fig. 4D). Moreover, both the seedling fresh weight and dry weight were also significantly reduced by silencing GhMAC3e (Fig. 4 E, F).

On the longitudinal section along the stem center of the epicotyl of the seedlings, it can be observed that after silencing GhMAC3e, the number of the cortical cells per unit length of stem was increased coupling with significantly reduced longitudinal length of each of them (Fig. 4 G, H). These observations suggest GhMAC3e is required for plant growth, especially for cell elongation.

To explore the physiological and molecular mechanisms underlying the arrested stem apical growth in GhMAC3e-silenced cotton seedlings, firstly, the auxin concentrations in stem tips were extracted and quantified, and the auxin distribution in the petiole of the top leaves was directly visualized using DR5-GFP reporter system. The results showed that silencing GhMAC3e significantly reduced auxin concentration in the stem tips of cotton seedlings, meanwhile, the green fluorescence in the petiole phloem of was dramatically impaired (Fig. 5A-C). These results suggest that both auxin biosynthesis in stem tips and auxin transportation between stem and young leaves through petiole phloem could be attenuated by the suppressed expression of GhMAC3e, thereupon then block cotton growth and biomass accumulation.

To explore the relationship between GhMAC3e, auxin, and cotton growth, we investigated the influences of silencing GhMAC3e on the expression of GhXTH16 (xyloglucan endo-glucan transglucosylase/hydrolase 16), GhGH3.6 (Gretchen Hagen3.6), and GhSAUR12 (small auxin up RNA 12), which plays a crucial role in plant cell wall extension, auxin inactivation, and auxin responsive cell elongation, respectively. Substantial upregulation of GhXTH16 and Gh3.6 expression and downregulation of GhSAUR12 were observed in GhMAC3e-silenced cotton (Fig. 5D). Our findings indicate that GhMAC3e is involved in homeostasis of active and inactive auxin, auxin signaling, and cell wall extension, which play important roles in plant growth.

Overexpression of GhMAC3e enhances disease resistance of Verticillium wilt in Arabidopsis

To further elucidate the function of GhMAC3e, the wild-type Col-0 Arabidopsis plants were genetically modified using Agrobacterium tumefaciens to create transgenic lines overexpressing GhMAC3e driven by the CaMV35S promoter. Among 17 independent transgenic lines, two pure lines, named 35S::GhMAC3e #23 and 35S::GhMAC3e #40, were selected for further analysis, because each line has one single transgene insertion and significantly elevated expression level of GhMAC3e (Fig. S2A, B). The 10-day-old transgenic and wild-type Arabidopsis plants (WT) were inoculated with VD strain Vd991 to evaluate VW disease resistance. At 10 days post-inoculation (dpi), the Arabidopsis leaves exhibited characteristic chlorosis, wilting and necrosis symptoms (Fig 6A). In comparison to the wild-type Arabidopsis plants, these symptoms were much weaker in overexpression transgenic lines coupling with significantly reduced fungal biomass (Fig. 6B), even though the typical disease symptoms became more pronounced in both the wild type and the transgenic lines as inoculation duration increased. So we examined the survival rates to evaluate durable resistance, the overexpression line 40# with the highest expression of GhMAC3e was selected for survival rate assessment. By 19 dpi, most leaves in the wild-type plants exhibited chlorosis and wilting, and even some individual plants died completely, in contrast, much fewer yellow leaves showed on the transgenic plants (Fig. 6C). The survival rate of the WT plants started to decline at 18 dpi and completely decreased to 0 at 27 dpi, while that of the transgenic plants began to decline at 22 dpi and decreased to 0 at 36 dpi. Overexpression of GhMAC3e can delay Arabidopsis death caused by infection with VD991 (Fig. 6D), thereby gaining more persistent tolerance to the fungus. Taken together, the ectopic overexpression of GhMAC3e conferred Arabidopsis enhanced resistance to VD. To explore the possible molecular mechanisms underlying this enhancement in VW resistance, the expression of AtNPR1, AtPR1, AtPR1, AtMYC2, AtVSP2, AtTGA2, AtERF1, and AtAAO3 of WT and the transgenic line 40# in response to VD infection (2 dpi) was analysed using RT-qPCR. As shown in Fig. 7, overexpression of GhMAC3e did not alter AtERF1 expression, implying that the advanced VW resistance may not employ ethylene-signaling regulation pathway. AtMYC2 and AtVSP2, the two important members in the same JA signaling branch, were down-regulated in the transgenic plants after VD inoculation; AtAAO3, a key gene in ABA biosynthesis pathway, was also down-regulated in transgenic plants both under regular and VD infection condition. The transgenic GhMAC3e can slightly decrease the expression level of AtNPR1 and AtTGA2 without VD infection, but did not influence their expressions after VD infection. Increased expression of the AtPR1 and AtPR2 genes marks the activation of SA signaling pathway, VD inoculation increased AtPR1 expression by more than 146 fold in WT, ectopic expression of GhMAC3e dramatically upregulated AtPR1 expression by around 15 fold and up to more than 262 fold under basal and VD inoculation condition, respectively. Whereas, AtPR2 expression was moderately induced by VD, but was compromised by overexpression of GhMAC3e. Together, ectopic expression of GhMAC3e seems to regulate SA rather than JA, ABA, ET, or reactive oxygen species (ROS) signaling in response to VD infection. Furthermore, GhMAC3e regulating SA signaling is independent upon GhNPR1 transcription level.

Plant materials and growth conditions

The cotton material used in this experiment was the standard country cotton genetic line, TM-1. It was cultivated in a greenhouse under conditions of 25°C, 80% relative humidity, and a photoperiod of 16 hours of light followed by 8 hours of darkness. Additionally, wild-type Columbia ecotype (Col-0) plants and transgenic Arabidopsis plants overexpressing GhMAC3e were grown in a separate greenhouse. The conditions for these plants were 22°C, 80% relative humidity, and the same 16-hour light/8-hour dark photoperiod.

Hormone treatment

In the hormone treatment experiments, the roots of two-week-old cotton seedlings were subjected to various treatments. These included spraying with sterile water as a control (mock), and with the following hormone solutions: Salicylic acid (SA) at 2.5 mmol/L, gibberellin (GA₃) at 100 µmol/L, abscisic acid (ABA) at 100 µmol/L, ethylene tetrachloride (ETH) at 2.5 mmol/L, and methyl jasmonate (MeJA) at 100 µmol/L. Root samples were collected at different time points post-treatment: 0 hours, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours. Immediately after collection, the samples were frozen in liquid nitrogen and subsequently stored at -80°C until analysis. Each hormone treatment was replicated three times, with each replicate comprising samples from three individual plants.

Virus-induced gene silencing assay

The specific fragment for the GhMAC3e gene was amplified from TM-1 cDNAs using PCR. This fragment was then inserted into the vector pTRV2 to create the construct TRV2:GhMAC3e. Subsequently, this construct was transformed into the Agrobacterium tumefaciens strain GV3101. To assess the efficiency of Virus-Induced Gene Silencing (VIGS), a control vector, TRV2:CLA1, was utilized. The VIGS procedure for cotton was according to that described by Gao et al(Gao, et al. 2011).

Arabidopsis transformation and screening of transgenic plants

The Coding Sequence (CDS) of the GhMAC3e gene was cloned into the pBI121 vector, which harbours the 35S promoter. Agrobacterium tumefaciens strain GV3101 was employed to transform the Arabidopsis ecotype Col-0 using the floral dip method(Clough and Bent 1998). Approximately seven weeks post-transformation, Arabidopsis seeds were harvested. To screen for positive transgenic lines, the seeds from the infiltrated plants were selected using 1/2 MS medium supplemented with 50 mg/L kanamycin. T₃ homozygous lines of the transgenic plants were then chosen for further experiments. The presence of gene insertion and the expression level were confirmed by PCR using genomic DNA as templates, and by quantitative real-time PCR, respectively.

Gene expression analysis

Total RNAs were extracted from cotton and Arabidopsis tissues using the RNAprep Pure Plant Kit (TIANGEN, China). The quality of the RNA was assessed by agarose gel electrophoresis, and only DNA-free RNA samples were used for subsequent analyses. For each sample, 500 ng of total RNA was used to synthesize cDNA with Superscript III reverse transcriptase (Invitrogen, San Diego, USA). RT-PCR and quantitative real-time PCR (qRT-PCR) analyses were then performed. The qRT-PCR reactions were carried out on the 7500 Real Time PCR System (ABI, Foster City, USA) with SYBR green (Bio-Rad, USA) as the fluorescent dye. For cotton, GhACT9 (AY305737) was used as the endogenous reference gene, while UBQ10 was used for Arabidopsis. The relative transcript levels were determined, normalized to the reference gene levels, and averaged over three biological replicates.

Culturing of V. dahliae, infection, and disease scoring

Vd991, a defoliating isolate of V. dahliae, was cultured on potato dextrose agar (PDA) at 25 °C for 4 d, and then inoculated into Czapek liquid medium (0.3% NaNO₃, 0.1% K₂HPO₄, 0.05% MgSO₄⋅7H₂O, 0.05% KCl, 0.001% FeSO₄, 3% sucrose, pH 7.2) for another 4d, the suspension was harvested and adjusted to 106 conidia/mL before further use. Arabidopsis plants were grown for 3 weeks before the roots were incubated in spore suspensions for 3 min, and subsequently transplanted into autoclaved vermiculite.

The survival rate was counted from the first dead Arabidopsis plant until all Arabidopsis plants died (the death was based on the yellow disease in the top leaf of the most rosette leaf and no further differentiation into new leaves).

After 10 days of inoculation, remove the leaves from the same position, disinfect and grind the leaves into a homogenate. Fungal biomass in plant tissues was measured using RT-PCR with primers specific for V. dahliae (ITS1-F and STVe1-R).

Identification of auxin content and distribution in cotton

For the determination of auxin content: weigh a unit weight of plant tissue, grinding it in liquid nitrogen, and extracting it with a mixture of propanol, water, and hydrochloric acid. After centrifugation and supernatant collection, the sample is reconstituted with 80% methanol and passed through a C18 column. The eluate is dried, dissolved, and filtered before LC-ESI-MS analysis using a 5 μm C18 column, with mobile phases of methanol and 0.05% formic acid, at a flow rate of 300 μL/min and column temperature of 30℃.

A tool commonly used in plants to detect distribution of plant auxin is a series of reporter genes based on the DR5 promoter. The DR5 promoter acts as an output of plant auxin signalling and its activity correlates with the distribution of auxin. We used ImageJ software to isolate the green channel in the photographs and observe the distribution of auxin. Agrobacterium tumefaciens with activated DR5-GFP growth factor indicator vector was shaken to OD600=0.6, and centrifuged at 4℃, 4000rpm for 10min; the bacteria were resuspended to OD600=0.6 with transient infiltration resuspension solution (250 mg of glucose, 5 ml of 500 Mm MES, 5ml of 20 mM NaPO₄.12H₂O, and 5 μl of 1 M AS to 50 ml), incubated in the dark at room temperature for 3 h, and injected into cotton cotyledons. 7 days later, cotton stems were sliced and observed using a macro zoom somatic vision microscope.

In this study, we explored the function of GhMAC3e, a homologue of MAC3A/B in cotton, focusing on its role in plant growth and resistance to VD, a vascular pathogen causing Verticillium wilt. This disease affects a broad range of globally significant crops, including cotton, leading to substantial economic losses. Our findings indicate that GhMAC3e plays a significant role in both plant growth and defense responses against VD. Overexpression of GhMAC3e in cotton enhances resistance to this pathogen. Conversely, silencing GhMAC3e inhibits the elongation of the main stem and reduces biomass accumulation. These results contribute to our understanding of the functions of MAC core components in plant growth and responses to fungal pathogen infections.

Previous studies have demonstrated that MAC3A and MAC3B are involved in the global regulation of splicing in plants(Jia, et al. 2017). Given its high homology with MAC3A/B, GhMAC3e may possess similar functions. GhMAC3e is expressed ubiquitously across various cotton tissues, and its expression is induced by salt, PEG stress, and VD infection. The application of exogenous hormones, including ABA, ETH, GA₃, MeJA, and SA, significantly induces the expression of GhMAC3e to varying degrees and at different times. These findings suggest that GhMAC3e could be involved in stress response and hormone signaling pathways, thereby regulating processes related to plant growth, development, and stress tolerance.

MAC3A/B were initially identified as interacting partners of MOS4 and play redundant roles in plant innate immunity. Double mac3a mac3b mutant plants exhibit a dwarfing phenotype(Monaghan, et al. 2009) . Previous research has demonstrated that auxin enhances the interaction between MAC3A/B and the transcription factor ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR13 (ERF13) by facilitating MPK14-mediated ERF13 phosphorylation, thereby promoting lateral root (LR) emergence(Yu, et al. 2024). Additionally, MAC3A/3B positively control hypocotyl growth through co-condensation with photoexcited cryptochromes in Arabidopsis (Jiang, et al. 2023). Based on these findings, we speculate that GhMAC3e may be involved in growth regulation in cotton. In our study, the silencing of GhMAC3e in cotton resulted in the inhibition of primary stem elongation and a reduction in biomass accumulation. These phenotypic changes are likely due to the decreased auxin content in the petioles and phloem of cotton true leaves. We have also observed that the expression levels of early auxin response genes GhXTH16, Gh3.6, and GhSAUR12 in GhMAC3e-silenced plants are significantly different from those in control plants. These genes are crucial in auxin signaling and mediate the regulation of various physiological processes by auxin. Specifically, GhXTH16 is involved in the differentiation of nutritive and reproductive meristematic tissues, as well as in the cell elongation of inflorescences and stems(Shao, et al. 2011). Gh3.6 encodes an IAA-amidohydrolase that interacts with auxin and amino acids alanine (Ala), aspartate (Asp), phenylalanine (Phe), and tryptophan (Trp), catalyzing the formation of amino acid conjugates with IAA(Staswick, et al. 2005; Sun, et al. 2019; Yu, et al. 2018). GhSAUR12 is rapidly induced by auxin and has been used as a reporter gene to investigate auxin-related mutants(Li, et al. 2017; Sun, et al. 2019). In conclusion, GhMAC3e modulates plant growth by altering auxin content, which may be mediated through changes in the expression levels of GhXTH16, Gh3.6, and GhSAUR12. However, the precise regulatory mechanisms underlying these interactions require further elucidation.

Verticillium wilt is a highly destructive fungal disease affecting a wide range of plants, significantly reducing the quality and yield of cotton. Understanding the molecular mechanisms underlying cotton resistance to VD is crucial for identifying valuable genes and developing wilt-resistant cotton varieties. In this study, we found that overexpression of GhMAC3e enhances disease resistance to Verticillium wilt. Concurrently, in comparison with controls, the expression level of AtPR1 is tremendously upregulated, while the expression levels of AtNPR1 and AtTGA2 were unaffected by the ectopic GhMAC3e in Arabidopsis after VD infection. These findings suggest that GhMAC3e is involved in the regulation of SA signaling pathways . In Arabidopsis, the TGA2-NPR1-NIMIN complex inhibits the expression of PR1 in the absence of SA, while in the presence of SA, SA induces a conformational change in NPR1, leading to the formation of a TGA2-NPR1-TGA3 activator complex that binds to the PR1 promoter, thereby activating PR1 expression. MAC3A/B can interact with TGA2, as MAC3A/B possess E3 ubiquitin ligase activity, which allow the degradation of TGA2 via ubiquitination. If so, the TGA2-dependent expression of PR1 would be arrested by overexpression of GhMAC3e, this is completely opposite to our experimental results. Previous studies have shown that NPR1 can form a ternary complex with TGA2 and inhibit PR1 expression through interaction with the transcriptional repressor TOPLESS, and SA treatment induces a conformational change in NPR1, leading to the dissociation of the TGA2-NPR1 complex and the formation of an activating complex, which in turn activates PR1 expression(Choi, et al. 2010; Seyfferth and Tsuda 2014), it is worth further studying whether and how MAC3 participates in this process.

Author contributions

WL, WJ and PT performed the experiments; QY and HZ analyzed the data; HZ drafted the paper; LK conceived and designed the experiments, revised the paper. All authors read and approved the final manuscript.

Funding

This work is supported by the National Natural Science Foundation of China (No. 31371672 and No. 32172083).

Conflict of interest The authors declare that they have no conflict of interests.

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FigS1.pdf
Fig.S1 Identification of virus induced silencing of GhMAC3e cotton. (A) plants infiltrated with TRV2:GhCLA1 displayed the photobleaching phenotype. (B) Transcription level of GhMAC3e in leaves of control plants and silenced plants was detected by RT-PCR
FigS2.pdf
Fig.S2 Expression analysis of GhMAC3e in transgenic Arabidopsis by RT-PCR or qRT-PCR

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Journal Publication

published 10 Oct, 2024

Read the published version in Plant Cell Reports →

Editorial decision: Minor revisions
17 Sep, 2024
Reviewers agreed at journal
20 Aug, 2024
Reviewers invited by journal
20 Aug, 2024
First submitted to journal
13 Aug, 2024
Editor assigned by journal
07 Aug, 2024

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GhMAC3e is involved in plant growth and defense response to Verticillium dahliae

Status:

Journal Publication

Version 1

Abstract

Figures

Key message

Introduction

Results

MAC3 protein analogs in upland cotton genome

Expression pattern of GhMAC3e

Silencing GhMAC3e compromised primary stem growth and biomass accumulation in cotton

Overexpression of GhMAC3e enhances disease resistance of Verticillium wilt in Arabidopsis

Materials and methods

Plant materials and growth conditions

Hormone treatment

Virus-induced gene silencing assay

Arabidopsis transformation and screening of transgenic plants

Gene expression analysis

Culturing of V. dahliae, infection, and disease scoring

Identification of auxin content and distribution in cotton

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1