The Arabidopsis thaliana Col-0 ecotype was used in this study. Arabidopsis plants were grown in a growth chamber at 22°C, with 10 h-day/14 h-night photoperiod and 100 μmol m⁻² s⁻¹ light intensity. Mutants mpk3-1 (Salk_151594), mpk6-2 (Salk_073907), and acs2/6/4/5/9 (CS16644) were obtained from the Arabidopsis Biological Resource Center (ABRC). MPK6SR (mpk3 mpk6 P_MPK6:MPK6^YG), MKK4-DD, NahG, and mkk4/mkk5 double mutants were kindly provided by Dr. Suqun Zhang (University of Missouri, MO, USA). To generate MKK5-DD and MEK2-DD transgenic lines, Arabidopsis MKK5 and tobacco NtMEK2 complementary DNAs (cDNAs) were cloned as described in previous studies (Yang et al., 2001; Ren et al., 2002). The mutant cDNAs with a FLAG epitope at their N-termini were subcloned into the steroid-inducible pTA7002 binary vector (Aoyama and Chua, 1997). Then, the constructs were transferred into GV3101 to be transformed into Col-0 plants using the floral-dip method.

For gene expression analysis, Arabidopsis was cultured in half-strength Murashige and Skoog (1/2 MS) liquid medium for 10 days. The resulting 10-day-old seedlings were mock treated (1/2 MS liquid medium only) or treated with 2 × 10⁸ cfu/ml GV3101 agrobacteria cells suspended in 1/2 MS liquid medium without shaking. Samples were harvested at four time points (3, 6, 12, and 24 h postinoculation (hpi) with agrobacteria). Three replicates were performed for each treatment. The seedlings were harvested at each time point and immediately frozen in liquid nitrogen; within each replicates, all seedlings were pooled together for RNA extraction and quantitative real-time PCR (qRT-PCR) analysis.

Total RNA was extracted from infected Arabidopsis seedlings using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from the extracted messenger RNA (mRNA) using oligo dT primer and reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed using a Bio-Rad CFX96 Real-Time System and iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) with 40 cycles. Templates were normalized with Arabidopsis UBQ10. Three biological and technical replicates were performed. Data were analyzed using the BioRad CFX Manager 2.0 Software. The comparative cycle threshold method (ΔΔCt) was used to obtain the relative fold change. Primer information is provided in Supplementary Table 1.

For RNA-sequence (RNA-seq) analysis, 10-day-old Arabidopsis seedlings of Col-0 and mkk4/5 were mock treated or treated with agrobacteria for 24 h. The seedlings were collected individually and frozen immediately in liquid nitrogen for RNA-seq. Three biological replicates were performed. Total RNA was used to make RNA-seq libraries with the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) following the recommendations of the manufacturer. These libraries were sequenced by Beijing Genomics Institute (BGI, Shenzhen, China).

The clean reads of all RNA-seq data obtained from BGI were mapped to A. thaliana TAIR10 genome by hisat2 (version 2.1.0) with default parameters. However, the above mapped SAM files were sorted and indexed by SAMtools (version 1.5). The expression data were calculated using featureCounts (v1.6.1) in default setting (Li et al., 2009; Liao et al., 2014; Kim et al., 2019). Genes with at least two-fold changes of expression in agrobacteria treatment compared to the mock treatment were identified as agrobacteria-responsive genes (ARGs). ARGs that were no longer induced by agrobacteria in mkk4/5 were identified as MKK4/MKK5-dependent ARGs.

To evaluate transformation frequency in whole Arabidopsis seedlings, the AGROBEST infection assay was used (Wu et al., 2014). Ten-day-old Arabidopsis seedlings grown in 1/2 MS liquid medium were infected with 10⁸ cfu/ml GV3101-pBISN1. At 3-day postinfection (dpi), seedlings were harvested for histochemical β-glucuronidase (GUS) staining and activity testing.

The A. tumefaciens tumorigenic strain A208 was used for stable transformation assays. Roots from 14-day-old Arabidopsis plants grown in sterile square Petri dishes containing sterile Gamborg's B5 medium were cut into ~5-mm segments. Root segments were assayed as described by Tenea et al. (2009). Tumors were scored after 30-day cultivation on MS medium. Three replicates were performed for each experiment. Statistical analysis was performed using Student's t-test.

For histochemical GUS staining, infected seedlings were stained in 0.2 mg/L 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) solution at 37°C overnight and then destained with 70% ethanol for observation. β-galactosidase activity was assessed with a fluorescent substrate (4-methylumbelliferyl-β-D-glucuronide (MUG assay)) to quantitatively determine GUS activity. These assays were performed according to the method of Jefferson et al. (1987). All of these were detected at 3 days after infection. Three replicates were performed.

Seedlings cultured for 10 days in liquid 1/2 MS medium were used for protein extraction. These 10-day-old seedlings were mock treated (1/2 MS liquid medium only) or treated with agrobacteria cells suspended in 1/2 MS liquid medium. Samples were harvested at four time points (5, 15, 30, and 60 min). Total protein was extracted from the whole seedlings in an extraction buffer [50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 10 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, 10% glycerol, and 1 × protease inhibitor cocktail (Sigma, St. Louis, MO, USA)]. The concentration of each protein extract was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard.

For the immunoblot analysis, anti-FLAG (F3165, Sigma, St. Louis, MO, USA) and anti-pTEpY (#4696, Cell Signaling Technology, Danvers, MA, USA) antibodies were used to detect MKK4-DD, MKK5-DD, and NtMEK2-DD protein expression and MPK3/MPK6 activation, respectively.

The leaf infiltration of Arabidopsis with Agrobacterium GV3101 carrying pBISN1 (GV3101-pBISN1) Arabidopsis was performed as described previously (Wroblewski et al., 2005). Agrobacteria cells resuspended in an infiltration buffer (10 mM MES, 10 mM MgCl₂, and 100 μM acetosyringone at pH 5.6) were injected into 4-week-old plant leaves. Then, the leaves were used for H₂O₂ detection and cell death analysis.

H₂O₂ was detected by the 3,3′-diaminobezidine (DAB) uptake method, which is an endogenous peroxidase-dependent in situ histochemical staining procedure using DAB (Thordal-Christensen et al., 1997). Leaves that were infiltrated with agrobacteria or mock infiltrated and treated with dexamethasone (DEX) were detached and placed in DAB solution (1 mg/ml, pH 5.5) for 2 h. The leaves were then boiled in ethanol (96%) for 10 min and then stored in 96% ethanol. H₂O₂ production is visualized as a reddish-brown coloration.

Ten-day-old Arabidopsis seedlings were infected with agrobacteria cells suspended in 1/2 MS liquid medium (2 × 10⁸ cfu/ml) or mock infected (1/2 MS liquid medium only). Samples were harvested at 24 h after inoculation and frozen in liquid nitrogen. Approximately 100 mg of each sample was suspended in 90% (V/V) methanol at a ratio of 1:1000. Samples were vortexed, sonicated for 15 min, and centrifuged at 15,000 × g for 20 min two times. The supernatant was transferred to a tube, and the SA solution was filtered and separated on a C18 analytical column (Waters, Milford, MA, USA) using high performance liquid chromatography (HPLC) and detected using fluorescence (excitation at 305 nm, emission at 405 nm; Waters, Milford, MA, USA). The mobile phase used for HPLC was 90% methanol and 0.1% formic acid with a flow rate of 0.8 ml/min.

All data were analyzed using Student's t-test.

The activation of mitogen-activated protein kinases (MAPKs) and the induction of defense-responsive gene expression are the early steps in the immune response after plants sense an invading pathogen (Hammond-Kosack and Jones, 1996; Zhang and Klessig, 2001). To elucidate the plant immune response to Agrobacterium, currently we used Arabidopsis model to profile MPK3/MPK6 activation and defense-responsive gene expression during the Agrobacterium infection process. In this system, 10-day-old Arabidopsis seedlings of wild-type (WT) (Col-0) cultured in liquid medium were infected with the disarmed hyper-virulent Agrobacterium strain GV3101. Samples were harvested at various postinfection time points. Then, MPK3/MPK6 activity and defense-responsive gene expression were assessed. The results showed that, compared with mock treatment, agrobacteria infection triggered a strong and rapid activation of MPK3/MPK6 (Figure 1A). During the agrobacteria infection process, the MPK3 and MPK6 activity reached peak levels at as early as 15 min postinfection and then decreased dynamically over time (Figure 1A). Meanwhile, after mock treatment, the MPK3 and MPK6 activity remained at a stable and low level during the whole stage, which was much lower than the level after inoculation with agrobacteria and showed no changes during the whole process (Figure 1A). Interestingly, we also observed other bands besides MPK3/MPK6 after agrobacteria infection. We inferred that another MAPK MPK4 is potentially activated by agrobacteria (Figure 1A). This finding demonstrates that MPK3/MPK6 signaling is involved in the process through which plants sense agrobacteria invasion. The expression of defense-responsive genes, such as FRK1, NHL10, At2g17740, and CRK11, was measured during the agrobacteria infection process. The results showed that the expression of these genes was strongly induced by agrobacteria inoculation, especially at the early stages (e.g., 3 and 6 h postinfection), but as infection time progressed, this induction faded somewhat (Figures 1B–E). In the mock treatment, the expression of these genes remained at very low levels, with no changes during the treatment process (Figures 1B–E). Together, these results demonstrate that MPK3/MPK6 activity and defense-responsive gene expression resulted from agrobacteria infection. Thus, we conclude that Agrobacterium can trigger plant immune responses, characterized by a rapid activation of MPK3/MPK6 and the induction of defense-responsive gene expression.

Previous studies have demonstrated that MKK4 and MKK5 are the upstream MAPKKs of MPK3/MPK6 that regulate the plant defense response and root development (Asai et al., 2002; Su et al., 2017; Shao et al., 2020). Because agrobacteria infection leads to a rapid and strong MPK3/MPK6 activation, we investigated whether MKK4 and MKK5 are required for the plant immune response to Agrobacterium. Here, we used mkk4/mkk5 double mutants generated from previously reported mkk4 and mkk5 single mutants (Zhao et al., 2014; Su et al., 2017) to determine the functions of these two MAPKKs in Agrobacterium-triggered immunity. After the inoculation of 10-day-old WT (Col-0) and mkk4/5 with the agrobacteria strain GV3101, samples were collected at various time points. MPK3/MPK6 activity and defense-responsive gene expression were measured. The results indicated that MPK3/MPK6 activity was induced and reached a high level at 15 min after the inoculation with agrobacteria in WT Col-0. However, this activity was abolished in mkk4/mkk5 double mutants (Figure 2A). Compared with the WT, MPK3/MPK6 activity induced by agrobacteria was impaired in mkk4/mkk5 double mutants (Figure 2A). Although some MPK3/MPK6 activities remained in mkk4/5 double mutants, it was much weaker than in WT (Figure 2A). These results demonstrate that MKK4/MKK5 are required for the induction of MPK3/MPK6 activity by agrobacteria and act upstream of MPK3/MPK6.

Meanwhile, our results revealed that the expression of some defense-responsive genes, such as FRK1, NHL10, At2g17740, and CRK11, was significantly impaired in mkk4/5 double mutants during agrobacteria infection, whereas the expression of those genes was strongly induced in WT, as indicated above (Figures 2B–E). Together, these results indicate that MPK3/MPK6 activity and defense-responsive gene expression induced by agrobacteria are highly dependent on the MKK4 and MKK5 function, and MKK4 and MKK5 are essential to Agrobacterium-triggered immunity.

Impaired plant immunity has been found to lead to increased transformation by Agrobacterium (Zipfel et al., 2006). As we have established that plant immune responses to agrobacteria are impaired in the mkk4/5 double mutants, we hypothesized that the mkk4/5 double mutant will exhibit a phenotype of enhanced Agrobacterium-mediated transformation. Hence, we employed two different transformation systems, namely transient (AGROBEST) and stable transformation (tumorigenesis induced by agrobacteria), to evaluate how the impaired immunity of this mutant affects the transformation. Firstly, we evaluated the transient transformation frequency in the whole seedling using the AGROBEST infection method (Wu et al., 2014). Ten-day-old seedlings of WT (Col-0) and mkk4/5 double mutants cultured in liquid medium were infected with the hyper-virulent nontumorigenic Agrobacterium strain GV3101 containing the binary vector pBISN1-GUS, which carries a GUS intron gene allowing its expression only in plants but not in Agrobacterium (GV3101-pBISN1). Then, GUS activity was assessed to monitor the transient expression efficiency at 3 dpi. Compared with WT plants, stronger and more concentrated spots of GUS staining were detected in mkk4/5 double-mutant seedlings at 3 dpi (Figure 3A). The enzymatic activity of GUS was ~1.8-fold greater in mkk4/5 double mutants than in WT (Figure 3A). Moreover, stable transformation via tumorigenesis was tested to confirm the function of MKK4/MKK5 in transformation. Root segments were infected with the tumorigenic A. tumefaciens strain A208 (Nam et al., 1997; Shi et al., 2014), and the root tumorigenesis efficiency was analyzed. Our results showed that compared to the WT, the mkk4/5 mutant exhibited ~1.8-fold greater tumorigenesis efficiency (Figure 3B), supporting the hypothesis that MKK4/MKK5 signaling is an important pathway in Agrobacterium-mediated transformation via the regulation of plant immunity.

Previous studies have demonstrated that gain-of-function activation of MKK4 or MKK5 were effectively inducing the plant defense responses in tobacco and Arabidopsis using transgenic lines with DEX-inducible MKK4 and MKK5 activities, respectively (Asai et al., 2002; Ren et al., 2002). To further elucidate the functions of MKK4 and MKK5 in the plant immune response to Agrobacterium and transformation, we analyzed the transgenic lines with DEX-inducible activity of both MKK4 and MKK5, designated as MKK4-DD and MKK5-DD, in which MKK4/MKK5 gene expression was induced by DEX. Firstly, we tested MPK3/MPK6 activation in DEX-inducible MKK4-DD and MKK5-DD transgenic lines. The results showed a strong MPK3/MPK6 activation in MKK4-DD and MKK5-DD transgenic lines after DEX treatment (Figure 4A), indicating that active MKK4/MKK5 can lead to the activation of MPK3/MPK6 and confirming that MKK4/MKK5 function upstream of MPK3/MPK6. The expression of defense-responsive genes, such as CRK11 and NHL10, was also tested in the MKK4-DD and MKK5-DD transgenic lines. After DEX treatment, CRK11 and NHL10 expression was dramatically induced in the MKK4-DD and MKK5-DD transgenic lines compared with in the WT, whereas DEX itself had no direct effect on the expression of these two genes in the WT (Figure 4B), indicating that DEX-induced defense-responsive gene expression in MKK4-DD and MKK5-DD plants resulted from MKK4/MKK5 activation.

Meanwhile, we assessed the Agrobacterium-mediated transformation frequency of the MKK4-DD and MKK5-DD transgenic lines. Using the AGROBEST method, WT (Col-0), MKK4-DD, and MKK5-DD transgenic plants were infected with GV3101-pBISN1. The GUS staining results showed that in DEX-treated samples, MKK4-DD and MKK5-DD plants exhibited a much lower transformation frequency at 3 dpi (Figure 4C). The measurement of GUS enzymatic activity showed that MKK4-DD and MKK5-DD plants had ~two-fold lower levels than WT after DEX treatment (Figure 4C). DEX had no effect on transformation frequency in WT (Figure 4C), demonstrating that the DEX-induced reduction of transformation frequency in MKK4-DD and MKK5-DD plants resulted from increased MKK4/MKK5 activation. Additionally, the tumorigenesis analysis was used to evaluate the stable transformation frequency of MKK4-DD and MKK5-DD transgenic plants. The tumorigenesis analysis showed the lower transformation frequency in MKK4-DD and MKK5-DD plants after DEX treatment, with fewer tumors in roots compared with WT (Figure 4D). Together, these results indicate that the activation of MKK4/MKK5 represses the transformation by agrobacteria, which may be due to the increases in MPK3/MPK6 activity and plant immunity.

Tobacco NtMEK2 is a homolog of Arabidopsis MKK4 and MKK5 (Ren et al., 2002). Previous research demonstrated that after DEX treatment, gain-of-function GVG-NtMEK2^DD transgenic Arabidopsis had high activation levels of MPK3 and MPK6 (Su et al., 2017), showing similar results to the MPK3/MPK6 activation pattern in MKK4-DD and MKK5-DD plants. As a result, we inferred that NtMEK2-DD transgenic Arabidopsis showed a similar response to agrobacteria. Unsurprisingly, when treated with DEX, NtMEK2-DD transgenic plants exhibited strong MPK3 and MPK6 activities (Supplementary Figure 1). After DEX treatment, NtMEK2-DD transgenic plants had much higher expression levels of CRK11 and NHL10 compared with WT, and DEX alone had no effect on WT (Supplementary Figure 1).

Similarly, transformation frequency was evaluated in NtMEK2-DD transgenic seedlings. In 3 days after inoculation with GV3101-pBISN1, the seedlings were stained in X-Gluc solution overnight. GUS staining appeared in more areas in WT (Col-0) seedlings than in NtMEK2-DD transgenic seedlings after DEX treatment (Supplementary Figure 1). The detection of GUS activity indicated 2.3-fold lower activity in NtMEK2-DD transgenic seedlings compared with the WT (Col-0) after DEX induction (Supplementary Figure 1). GUS staining and activity showed no changes in WT (Col-0) with DEX treatment, indicating that the lower transformation frequency in NtMEK2-DD transgenic seedlings resulted from DEX-induced MKK4/MKK5 activation.

Together, these results support the conclusion that MKK4/MKK5 and their homolog NtMEK2 play a positive role in plant immunity to Agrobacterium by activating MPK3/MPK6. The activation of MPK3/MPK6 and of downstream defense pathways are the important processes in the repression of transformation by Agrobacterium.

MKK4 and MKK5 are the upstream MAPKKs of MPK3/MPK6 in the regulation of plant defense responses (Asai et al., 2002; Su et al., 2017), and our data demonstrate that MKK4 and MKK5 also function upstream of MPK3/MPK6 in Agrobacterium-triggered immunity. Based on these results, we investigated whether these two MAPKs MPK3/MPK6 also regulate plant transformation by Agrobacterium. Our results reveal no significant transformation difference in mpk3 and mpk6 single mutants compared with the WT after inoculation with Agrobacterium GV3103-pBISN1 (Supplementary Figure 2), indicating that functional redundancy between MPK3 and MPK6 may exist in Agrobacterium-mediated transformation. As a result, a conditional mpk3/mpk6 double-mutant, MPK6SR, which is sensitive to NA-PP1 (a kinase inhibitor) (Su et al., 2017; Shao et al., 2020), was used for the analysis of Agrobacterium-mediated transformation in Arabidopsis. NA-PP1 makes the activity of MPK6SR plants nulls that are equivalent to mpk3/mpk6 double mutants (Xu et al., 2014; Su et al., 2017; Shao et al., 2020). Using this genetic material, we tested the frequency of transformation by Agrobacterium. After inoculation with GV3101-pBISN1, GUS staining and activity were detected in WT and MPK6SR seedlings. Compared with the WT, MPK6SR seedlings exhibited a higher transformation frequency, with a stronger GUS staining, and a 1.7-fold increase in GUS activity after treatment with NA-PP1 (Figure 5A). In the absence of the inhibitor NA-PP1, MPK6SR seedlings behaved like WT control (Figure 5A). Moreover, NA-PP1 itself had no effect on the transformation in WT (Col-0), demonstrating the specificity of NA-PP1 in plant transformation to MPK6SR lines.

We confirmed this result through the tumorigenesis analysis. As shown in Figure 5B, compared with WT, NA-PP1-treated MPK6SR roots had a much higher stable transformation frequency (Col-0), with more tumors in the roots. NA-PP1 itself had no effect on tumorigenesis in WT (Col-0). These results indicate that MPK3 and MPK6 act downstream of MKK4 and MKK5 to regulate Agrobacterium-mediated transformation.

Throughout the Agrobacterium-mediated transformation process, the host plants exhibit dynamic gene expression patterns in response to Agrobacterium. Changes in host gene expression patterns throughout the transformation process have effects on transformation frequency. To clarify the mechanisms of MKK4/MKK5 in regulating the Agrobacterium-mediated transformation process, global transcriptome analysis was performed to compare WT and mkk4/5 double-mutant plants. After data analysis, genes were considered significantly differentially expressed if they had a false discovery rate of less than 0.05 and a change in expression between the two treatments of at least two-fold. Overall, 7,702 unique genes in WT (blue circle) and 7,810 (yellow circle) in mkk4/5 were differentially expressed in the agrobacteria treatment relative to the mock treatment (Figure 6A). Of these genes, 5,065 were differentially expressed between treatments in both WT and mkk4/5 (Figure 6A). In total, 3,905 unique genes were induced by agrobacteria treatment relative to the mock treatment in WT, of which 694 showed no expression changes in mkk4/5 plants between the agrobacterium and mock treatments (Figure 6B). This finding indicates that these 694 genes are regulated by MKK4/MKK5 in the agrobacteria infection process and may be the major factors involved in the regulation of transformation by MKK4/MKK5.

We employed the gene ontology (GO) analysis to categorize the 3,905 agrobacteria-induced genes in WT to investigate wide-scale changes in gene expression related to specific cellular and biological processes. Figure 6C shows the degree to which selected GO biological process categories were overrepresented. Categories including “defense response to bacteria,” “response to SA,” “response to hydrogen peroxide,” “cellular response to oxidative stress,” “cell death,” and “response to jasmonic acid” are overrepresented among agrobacteria-induced genes in WT (Figure 6C, Supplementary Figure 3, Supplementary Material 1), indicating that agrobacteria induce a broad range of plant immune responses. In contrast, 694 of these genes, which were no expression changes in mkk4/5 mutant plants, were also categorized. The categories “defense response to bacteria,” “response to SA,” “response to hydrogen peroxide,” and “innate immune response” are overrepresented among these genes showing no change in expression (Figure 6D, Supplementary Figure 4, Supplementary Material 2). Thus, most plant defense pathways induced by agrobacteria were abolished in mkk4/5 mutant plants, further confirming that MKK4/MKK5 signaling is indispensable in the plant immune response to agrobacteria. Together, these results demonstrate that agrobacteria infection generally induces plant stress and defense response pathways, which are dependent on MKK4/MKK5 activity.

The production of ROS and cell death are active defense mechanisms of plants against invading pathogens. Our global transcriptome data indicated that the “response to hydrogen peroxide,” “cellular response to oxidative stress,” and “cell death” pathways were induced during the agrobacteria infection process, indicating that plants produced ROS and underwent cell death to counteract agrobacteria infection. To confirm the function of MKK4/MKK5 in Agrobacterium-mediated ROS production and cell death, leaves from a 4-week-old seedling of WT (Col-0), MKK4-DD, and MKK5-DD were inoculated with agrobacteria GV3101 through leaf infiltration, and then the H₂O₂ production and cell death were assessed. As shown in Figure 7A (upper layer), in the absence of agrobacteria, the reddish-brown precipitants of oxidized DAB, an indication of H₂O₂ production, were visible in the leaves of MKK4-DD, MKK5-DD transgenic plants pretreated with DEX as an inducer. As controls, the leaves of Col-0 plants were pretreated with DEX and processed alongside inoculated leaves, and no H₂O₂ generation was observed in these control leaves. The leaves from Col-0, MKK4-DD, and MKK5-DD transgenic plants clearly exhibited no H₂O₂ generation in the absence of DEX (Figure 7A, upper layer). However, in the presence of agrobacteria, the leaves of Col-0, MKK4-DD, and MKK5-DD transgenic plants all produced H₂O₂ (Figure 7A, lower layer). In this analysis, H₂O₂ production was also detected when the plants were pretreated with DEX, and the resulting H₂O₂ production was much greater in MKK4-DD and MKK5-DD transgenic plants than in plants treated only with DEX (Figure 7A), indicating that agrobacteria infection can induce ROS production in plants and that DEX-induced MKK4 and MKK5 activation promotes ROS production during the agrobacteria infection process.

Similarly, cell death during the agrobacteria infection process in Col-0, MKK4-DD, and MKK5-DD transgenic plants was investigated. As shown in Figure 7B, among the leaves of Col-0, MKK4-DD, and MKK5-DD transgenic plants pretreated with DEX for 1 day but not inoculated with agrobacteria, only MKK5-DD plants showed very low levels of cell death while all plants without DEX treatment had no cell death. In the presence of agrobacteria inoculation at 1 dpi or 1 day after DEX treatment, the leaves of Col-0, MKK4-DD, and MKK5-DD transgenic plants without DEX treatment had no cell death; however, after pretreatment with DEX, enhanced cell death was observed in MKK5-DD transgenic plants, with no cell death observed in Col-0, MKK4-DD plants (Figure 7B). At 2 dpi or 2 days after DEX treatment, in the absence of agrobacteria inoculation, the leaves from MKK4-DD and MKK5-DD transgenic plants pretreated with DEX showed higher levels of cell death compared to Col-0 plants, and no cell death occurred in plants without DEX treatment. However, in the presence of agrobacteria inoculation, cell death was enhanced in DEX pretreated MKK4-DD and MKK5-DD transgenic plant leaves compared to leaves without agrobacteria inoculation. As controls, no cell death was observed in Col-0, MKK4-DD, and MKK5-DD transgenic plants without DEX pretreated and agrobacteria inoculation. At 5 dpi or 5 days after DEX treatment, cell death was observed in MKK4-DD, and MKK5-DD transgenic plants when pretreated with DEX and in DEX pretreated agrobacteria-infiltrated leaves. As controls, agrobacteria-infiltrated Col-0, MKK4-DD, and MKK5-DD plants also exhibited clear cell death, whereas no cell death was observed in buffer-infiltrated Col-0, MKK4-DD, or MKK5-DD plants.

Based on these results, we conclude that agrobacteria can induce plant ROS production at an early infection stage (1 dpi) and cell death at a late infection stage (5 dpi), and that the activation of MKK4/MKK5 promotes agrobacteria-induced ROS production and cell death. Thus, ROS production and cell death are involved in plant immunity and regulated by MKK4/MKK5. This pathway may be one factor influencing Agrobacterium-mediated transformation.

Salicylic acid is an important compound in plant defense (Vlot et al., 2009). Previous research demonstrated that plants with constitutively active MPK3 plants accumulate SA (Geno et al., 2017). SA accumulated at 6 days after inoculation with agrobacteria (Lee et al., 2009). Our transcriptome data indicated that SA signaling is a plant defense response to Agrobacterium. To explore whether SA is involved in MKK4/MKK5-regulated Agrobacterium-mediated transformation, we first examined the expression of SA synthesis genes AtPBS3, AtICS1, AtEDS5 (Rekhter et al., 2019; Torrens-Spence et al., 2019) in the WT, and mkk4/5 double mutant. As shown in Figures 8A,B, AtPBS3 was induced after agrobacteria inoculation in the WT (Col-0), but this induction effect was impaired in mkk4/5 double mutants. AtICS1 and AtEDS5 gene expressions were induced by agrobacteria inoculation in the WT, but the expression of AtICS1 was higher in the mkk4/5 mutant than in WT and AtEDS5 expression showed no difference between mkk4/5 double mutants and the WT (Col-0) mutant (Supplementary Figure 5). We then measured SA levels in plants when inoculated with agrobacteria. Whole WT (Col-0) and mkk4/5 seedlings were harvested after inoculation with agrobacteria, and their SA contents were determined via HPLC. No significant difference between the WT and mkk4/5 was found in the absence of agrobacteria inoculation (Figure 8C). Agrobacteria inoculation induced SA accumulation in WT, but this induction was abolished in the mkk4/5 mutant (Figure 8C), indicating that the loss of the function of MKK4/MKK5 resulted in impaired SA induction by agrobacteria in Arabidopsis. These results demonstrate that MKK4/MKK5 regulate the agrobacteria induction of SA synthesis during the infection process.

As SA synthesis is regulated by MKK4/MKK5 during agrobacteria infection, we next analyzed the effect of exogenous SA on transformation in WT and mkk4/5 double mutants. The results showed that applying SA to WT (Col-0) and mkk4/5 double-mutant seedlings caused the transformation frequency of both the WT (Col-0) and mkk4/5 to decrease relative to plants without SA treatment, with ~1.6- and 1.8-fold decreases, respectively, in GUS activity (Supplementary Figure 5). This result demonstrates that exogenous SA rescues the phenotype of mkk4/5 and greater SA concentration represses agrobacteria infection in Agrobacterium-mediated transformation. To obtain more evidence supporting this hypothesis, we examined the transformation of SA-deficient mutant NahG plants (van Wees and Glazebrook, 2003). The results indicated that, at 3 days after inoculation with GV3101-pBISN1, GUS staining was much stronger in NahG seedlings than in WT (Col-0), with a GUS activity increase of 2.9-fold compared with the WT (Col-0) (Figure 8D). Stable transformation via tumorigenesis was also analyzed. WT and NahG root segments were infected with the tumorigenic A. tumefaciens strain A208, and tumorigenesis efficiency was counted. The data show that compared with WT, the NahG mutant exhibited a much more agrobacteria-sensitive phenotype and had more tumors in roots (Figure 8E). From these results, we can conclude that SA accumulation during agrobacteria infection is regulated by MKK4/MKK5 and is involved in Agrobacterium-mediated transformation.

As an important plant hormone, ET plays a major role in plant defense response to pathogen infection (Wang et al., 2002). MPK3/MPK6 regulate ET biosynthesis during pathogen attack (Han et al., 2010; Li et al., 2012). Agrobacterium has been found to regulate ET metabolism during the transformation process. Levels of the ET precursor ACC (1-amino-cyclopropane-1-carboxylate) were elevated during infection by agrobacteria (Lee et al., 2009). To clarify the roles of MKK4/MKK5 in regulatory pathways during the agrobacteria infection process, we investigated whether ET is involved in this process. Firstly, we measured the gene expression of rate-limiting enzyme of ET biosynthesis ACC synthetase (ACS) during agrobacteria infection. The results show that with agrobacteria inoculation, the ACC biosynthesis genes ACS2, ACS4, and ACS6 were strongly induced compared with the mock treatment. These genes expression levels peaked at 6 hpi and decreased with further time after inoculation (Figures 9A,B; Supplementary Figure 6). This finding indicates that plants increased ET synthesis during agrobacteria infection.

We also detected the expression of ACS genes in mkk4/5 double mutants. The results reveal that ACS2 and ACS4 gene expressions were impaired in mkk4/5 double mutants inoculated with agrobacteria at 3 hpi while ACS6 expression was impaired at 24 hpi (Figures 9C,D; Supplementary Figure 6). No difference in ACS6 expression occurred in the WT (Col-0) mutant or mkk4/5 double mutants at 3 hpi (Supplementary Figure 6), indicating that the regulation of ACS6 gene expression occurs late in the agrobacteria infection process. These results imply that ET synthesis is regulated by MKK4/MKK5 at the ACS gene transcription level in the agrobacteria infection process.

To further explore the relationship between MKK4/MKK5 signaling and ET during Agrobacterium-mediated transformation, we applied exogenous ACC to the WT mutant and mkk4/5 double mutants to determine whether ACC can rescue the transformation phenotype of mkk4/5 double mutants. After treatment with ACC, mkk4/5 double-mutant seedlings exhibited a two-fold decrease in the transformation frequency compared with the mock treatment (Supplementary Figure 6), indicating that the application of the exogenous ET precursor ACC can reverse the increased transformation phenotype of mkk4/5. Notably, ACC also caused the transformation frequency to decrease in the WT (Col-0) (Supplementary Figure 6), indicating that high ET levels had negative effects on the transformation by agrobacteria. To confirm the role of ET in transformation, we checked the transformation phenotype of ET synthesis defective mutant acs2/6/4/5/9. This mutant exhibited very low ET production compared with the WT (Col-0) (Li et al., 2012). Transient transformation frequency evaluation showed that acs2/6/4/5/9 mutant seedlings had a greater GUS staining and 2.1-fold higher GUS activity than WT (Col-0) (Figure 9E). Tumorigenesis was analyzed as described earlier. WT and acs2/6/4/5/9 mutant root segments were infected with the tumorigenic A. tumefaciens strain A208, and tumorigenesis efficiency was measured. The data show that the acs2/6/4/5/9 mutant had more tumors in roots and exhibited a higher transformation frequency than WT (Figure 9F). These results suggest that ET synthesis is regulated by the MKK4/MKK5 signaling pathway and is involved in the transformation by Agrobacterium.