Possible Involvement of MYB44-Mediated Stomatal Regulation in Systemic Resistance Induced by Penicillium simplicissimum GP17-2 in Arabidopsis

The plant growth-promoting fungus (PGPF), Penicillium simplicissimum GP17-2 (GP17-2), induces systemic resistance against Pseudomonas syringae pv. tomato DC3000 (Pst) in Arabidopsis thaliana. The molecular mechanisms underlying induced systemic resistance (ISR) by GP17-2 were investigated in the present study. Microscopic observations revealed that stomatal reopening by Pst was restricted by elicitation with the culture filtrate (CF) from GP17-2. A gene expression analysis of MYB44, which enhances abscisic acid signaling and consequently closes stomata, revealed that the gene was activated by CF. CF-elicited myb44 mutant plants failed to restrict stomatal reopening and showed lower resistance to Pst than wild-type plants. These results indicate that stomatal resistance by GP17-2 is mediated by the gene activation of MYB44. We herein revealed that the MYB44-mediated prevention of penetration through the stomata is one of the components responsible for GP17-2-elicited ISR.

Plants are surrounded by a large number of microorganisms, some of which improve plant adaptation to diverse stresses. Plant growth-promoting fungi (PGPF) are mutualistic microbes (24). We previously characterized the mutualistic activity of the PGPF isolate, Penicillium simplicissimum GP17-2 (GP17-2), which was isolated from the rhizosphere of zoysiagrass (Zoysia tenuifolia) (18,19). The culture filtrate (CF) from GP17-2 has the ability to elicit induced systemic resistance (ISR) in plants, effectively controlling several diseases such as anthracnose (13,27) and angular leaf spot in the cucumber (13), cucumber mosaic virus (CMV) in Arabidopsis and tobacco (4), and bacterial leaf speck in Arabidopsis (6).
Plant pathogenic bacteria invade plants through natural openings, such as stomata and water pores, or accidental wounds. Stomatal closure is an essential part of innate immunity against bacterial pathogens (2, 3, 14, 15, 20-22, 28, 31-33). In order to overcome stomatal closure by plants, Pseudomonas syringae pv. tomato DC3000 (Pst) forces stomatal reopening with the phytotoxin coronatine (COR) (34). Since the chemical structure of COR is similar to that of jasmonoyl-isoleucine (JA-Ile), COR has been suggested to activate JA signaling as an analog of JA-Ile in order to reopen stomata (20). A root treatment with abscisic acid (ABA) and salicylic acid (SA) was previously shown to restrict stomatal reopening by Pst (14), suggesting the involvement of ABAand/or SA-mediated signaling in ISR.
We have shown that SA-and jasmonate (JA)/ethylene (ET)-responsive genes were both activated by a root treatment with CF (6). Single mutants unable to transduce SA, JA, or ET signals alone still retained the ability to induce CF-elicited ISR (6). Based on these findings, we hypothe-sized that ISR is transduced by multiple signaling pathways in parallel, including the SA, JA, and ET pathways (6). We also observed that some ABA responsive genes, namely LEAs, RAB18, RD22, GOLS2, and ABI5, were activated by CF (data not shown). These findings prompted us to investigate whether ABA signaling is also involved in ISR.
The transcription factor, MYB44 (AT5G67300), has been identified as a stomata-specific enhancer of the ABA signal for stomatal closure (9). We were motivated to perform the study by the elicitor responsibility of MYB44 (8,16,24). We herein focused on MYB44-mediated stomatal resistance in order to identify downstream events mediated by CF.

Plant materials
The seeds of wild-type Arabidopsis thaliana (Col-0), a T-DNA knockout line of MYB44 (myb44, Salk_008606C), and overexpression line of MYB44 (MYB44ox, F19506) (7) were grown using the hydroponic culture system described by Toda et al. (29) for 2 weeks in all experiments. Briefly, Arabidopsis seeds were soaked in 0.5 mL of distilled water in 1.5-mL Eppendorf tubes, kept at 4°C for 2 d, and then placed on a nylon mesh (50 holes per inch) (Filter-net, Sansyo, Tokyo, Japan) held in a plastic photo slide mount (FujiFilm, Tokyo, Japan) (18 seeds per mount). The mounts were floated on 1/5 strength MGRL nutrients (pH 5.6) (5) and grown at 22°C under ~60 μE m -2 s -1 and 16/8-h (light/dark) conditions. The MGRL solution was renewed once a week after the start of the culture.

Culture of the fungus and bacterium
The PGPF P. simplissimum GP17-2 (GP17-2) isolated from the rhizosphere of zoysiagrass (Z. tenuifolia) (18,19) was maintained in potato dextrose agar (PDA) medium at 4°C and as a barley grain inoculum (BGI) at -20°C. In order to recover GP17-2, the BGI was placed on PDA medium in a 9-cm Petri dish and grown at 25°C for 7 d. The culture was then used to prepare CF.
The bacterial pathogen P. syringae pv. tomato DC3000 (Pst) was maintained as a 40% (v/v) glycerol stock at -80°C. In order to recover the Pst, the glycerol stock was cultured on a King's medium B (KB) plate with 5 μg mL -1 rifampicin at 25°C for 3 d. A single colony was selected and transferred to a 100-mL Erlenmeyer flask containing 50 mL of liquid King's medium B with 5 μg mL -1 rifampicin, and the culture was shaken at 130 rpm at 25°C for 2 d and then used for inoculation.

Preparation and treatment of CF
Twenty mycelial disks (8 mm in diameter) cut out from the colony margin on PDA were transferred to a 500-mL Erlenmeyer flask containing 200 mL of potato dextrose broth (PDB) and incubated without shaking at 25°C for 10 d in the dark. The culture broth was filtered through two layers of filter paper (Whatman qualitative filter paper No. 2, GE Healthcare Japan, Hino, Japan), and CF was then filter-sterilized using a 0.22-μm Millex-GV syringe filter unit (Merck Millipore, Darmstadt, Germany). The CF treatment was performed 1 d before the challenge inoculation by dipping the roots of 2-week-old plants into CF for 1 h. Control plants were treated with PDB or sterile distilled water (SDW) instead of CF.

Inoculation with the pathogen and disease assessment
CF-treated and PDB-treated plants were placed at 100% relative humidity for 23 h. Cultured bacterial cells, as described above, were collected by centrifugation at 3,000 rpm for 10 min, washed twice, and re-suspended with 10 mM MgSO 4 supplemented with 0.01% (v/v) Silwet L-77 (Nihon Unica, Tokyo, Japan) to a concentration with OD 600 0.07 (3.5×10 7 cfu mL -1 ). The challenge inoculation was performed by foliar spraying with the Pst suspension. Inoculated plants were incubated at 22°C and 100% relative humidity in the dark for 2 d and then under light for 1 d. The shoots were harvested, weighed, surface-sterilized twice by dipping in 100% Et-OH for 1 s, and immediately rinsed with sterilized distilled water. The shoots were then homogenized with a pestle in 1.5-mL Eppendorf tubes containing 1 mL of SDW, and 100-μL aliquots of the appropriate dilutions were then spread onto KB plates containing 50 μg mL -1 rifampicin. After a 48-h incubation at 25°C, Pst colonies (colonyforming units, cfu) were counted and determined as cfu gram -1 fresh weight of tissue. At least 3-6 replicates per treatment were analyzed. One replicate used six plants.

Measurement of the stomatal aperture
Two-week-old plants were treated with CF as described above. Similarly, plants were treated with 100 μM ABA, SDW, or PDB. Plants were inoculated 23 h after the treatments with Pst using the inoculum prepared, as described above, by shoot immersion and incubated at 22°C under light. Leaves were harvested before and 1 and 3 h after the inoculation, and the abaxial surface was immediately coated with collodion (Ekivan A, Taihei Medicine, Ibaraki, Japan) to take an impression of the stomata. The impression was observed under a light microscope (Olympus BX-70, Tokyo, Japan) at 400× magnification, and images of the stomata were acquired randomly from five leaves of five different plants. The widths of the stomata (n=60) were measured using Pix2000_Pro software (Inotech, Hiroshima, Japan). Experiments were performed in triplicate.

Gene expression analysis
Two-week-old plants treated with CF and PDB by the same method used for the measurement of the stomatal aperture following the inoculation were harvested for RNA extraction at the indicated time points. Samples were collected in 1.5-mL Eppendorf tubes and ground in liquid N 2 with a pestle. Total RNA was extracted using Sepasol ® -RNA I Super G (Nacalai Tesque, Kyoto, Japan) following the manufacturer's protocol. The concentrations of the extracted RNA were measured with a spectrophotometer (BioSpectrometer ® Basic; Eppendorf, Tokyo, Japan), and 500 ng of total RNA was used to synthesize first-strand cDNA by ReverTra Ace qPCR RT Master Mix with a gDNA Remover (TOYOBO, Osaka, Japan), following the manufacturer's protocol. The reverse transcription products (10 μL) were diluted by one half with sterile water and used as templates for real-time quantitative PCR (qRT-PCR) performed using SYBR ® Premix Ex Taq TM II (Tli RNaseH Plus) (TaKaRa, Otsu, Japan). qRT-PCR reaction mixtures were prepared in a total volume of 10 μL containing 5 μL of 2×SYBR Premix, 0.4 μL of 10 μM each of the forward and reverse primers (0.4 μM final concentration), 0.8 μL of the cDNA template, and 3.4 μL of dH 2 O. Gene-specific primers for MYB44 (AT5G67300) were forward 5ʹ-TCTCCACCTGTTGTTACTGGGCTT-3ʹ and reverse 5ʹ-TTGA CTCGTGGCTACGGTTTGACT-3ʹ (8) and for Actin2 (AT3G18780) as an internal control were forward 5ʹ-GGCAAGTCATCACGA TTGG-3ʹ and reverse 5ʹ-CAGCTTCCATTCCCACAAAC-3ʹ (17). The reactions were performed with a Thermal Cycler Dice ® Real Time System II (TP900, TaKaRa) under the following conditions: an initial denaturation step of 95°C for 30 s, 40 cycles of the twostep thermal cycling profile of denaturation at 95°C for 5 s, and primer annealing and extension at 60°C for 60 s. In order to verify the specificity of the amplicon for each primer pair, the final dissociation step was performed at 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. The relative standard curve method was used for the quantification of mRNA expression. cDNA standard curves were prepared using the threshold cycles with five serial dilution series (diluting cDNA samples to 1/2, 1/4, 1/8, 1/16, and 1/32). At least three technical replicates were analyzed for each sample. Five plants per condition were used.

Reduction in Pst resistance induced by CF in the myb44 mutant
The Pst population and symptoms in wild-type plants were significantly smaller and weaker, respectively, with the CF treatment (GP17-2) than with the control (PDB) (Fig. 1A and  B). In contrast, no significant difference was observed in the pathogen population in CF-treated myb44 mutants (Fig. 1C).

Stomatal reopening was restricted by CF and ABA
As shown in Fig. 2, the stomatal aperture of control plants (SDW) was reduced from before the inoculation (0 h) to 1 h after the inoculation, and closed stomata reopened 3 h after the inoculation. In CF-treated plants (GP17-2), the stomatal aperture was reduced from 0 to 1 h, a similar time frame to that required in control plants, and then reopened at 3 h, and was significantly weaker than that in control plants (decrease in the aperture size, 0.72-1.81 μm; decrease in open stomata, 10%-60% that of control plants). In ABA-treated plants (ABA), the stomatal aperture was significantly reduced without the inoculation (0 h), and reopening at 3 h was also markedly weaker than that in control plants (decrease in the aperture size, 0.52-1.10 μm; decrease in open stomata, 10%-20%), which was similar to that observed with the CF treatment (GP17-2). Fig. 3 shows that the treatment of Arabidopsis with CF increased the expression of MYB44 5.9-fold after 24 h, and the response observed was markedly stronger than that of the PDB control (Fig. 3, before inoculation). We then investigated the response of MYB44 to the inoculation of Pst after the CF treatment. As shown in the figure, subsequent mock treatments resulted in a small and transient induction 1 h after the inoculation in CF-treated and control plants (Mock in the  figure). The subsequent inoculation of Pst resulted in the

Requirement of MYB44 for stomatal closure by the CF treatment
The stomatal aperture was measured 3 h after the inoculation in CF-treated wild-type and myb44 mutant plants (Fig. 4). The stomatal aperture was smaller in CF-treated wild-type plants than in PDB-treated wild-type plants (decrease in the aperture size, 0.62-0.92 μm, decrease in open stomata, 10%-18%). In contrast, myb44 plants showed no reduction in the stomatal aperture by the CF treatment.
The stomatal aperture was smaller in MYB44 ox plants than in wild-type plants (decrease in the aperture size, 1.1 μm) without elicitation or pathogen recognition (Fig. 5).

Discussion
In our previous study, CF treatment-elicited ISR against Pst was identified in Arabidopsis (6). The restriction of stomatal reopening by Pst is an essential part of innate immunity against plant pathogenic bacteria (2, 3, 14, 15, 20-22, 28, 31-33). We found that CF-treated plants showed induced stomatal resistance against Pst (Fig. 2). The elicitor activity of CF has been detected in a dialysis fraction (>MW 12,000) (13). Since this activity did not disappear following a treatment with protease, autoclaving, or the removal of lipids, we concluded that the main elicitor compound is polysaccharide(s) (13). The same elicitor may trigger stomatal resistance against Pst. Since the primed state of stomatal resistance was sustained for 1 d, we hypothesized that CF-mediated stomatal resistance is achieved through transcriptional regulation. We focused on the transcription factor, MYB44 (AT5G67300) because it is a stomata-specific enhancer of ABA signaling for stomatal closure (9) and has been identified as an elicitor-responsive gene (16,25). These features led us to the hypothesis of the involvement of MYB44 in stomatal regulation by CF. We investigated the hypothesis that MYB44 mediates GP17-2elicited ISR by stomatal closure. The expression profiles (Fig.  3) and phenotypes of MYB44 mutants ( Fig. 1 and 4) supported our hypothesis, demonstrating that the gene activation of MYB44 for stomatal regulation is an essential part of ISR. One advantage of stomatal regulation by gene activation over rapid regulation at the protein level is the longer persistence of the regulated state of stomata, which supports the long  (Experiments 1-3). An asterisk denotes significant differences from corresponding controls based on the Student's t-test (P<0.05). memory of the priming effects of CF. myb44 mutant plants did not fail to close their stomata 1 h after the inoculation (data not shown), indicating that MYB44 is not essential for stomatal closure as innate immunity, but is important for supporting long-term stomatal resistance.
Although ISR by GP17-2 involves stomatal regulation, as discussed above, we do not exclude the additional possibility of the promotion of ISR, which involves the modulation of phytohormone effects by the pathogen infection. Pst invades plants by suppressing their SA-mediated defense responses through the activation of the JA pathway (34). In the present study, we identified the involvement of MYB44 in the process of GP17-2-elicited ISR (Fig. 1, 3, and 4). As reported previously, MYB44 enhances ABA signaling (9), and the ABA signal is known to be antagonistic to pathogen-associated JA signaling (1). Thus, the role of MYB44 in ISR may include antagonistic activity against JA signaling. MYB44 has also been shown to modulate SA signaling (26). The overexpression of MYB44 activates WRKY70 by directly binding to its promoter, thereby enhancing SA-mediated defense responses (26). Thus, the gene activation of MYB44 by CF may also activate SA-mediated defense responses. The relationship between GP17-2-elicited ISR and SA responses currently remains unknown.
Several studies have investigated the gene activation of MYB44, and showed that it is activated by environmental stresses (dehydration, low temperature, salinity, and wounding), pathogenic elicitors (flg22 and harpin protein [HrpN Ea ]), and stress-associated hormones (methyl jasmonate and ABA) (8-10, 16, 25, 30). It has not yet been established whether an unidentified elicitor in CF directly activates MYB44 or if gene activation is achieved via modulation by an unidentified phytohormone.
The treatment of plants with elicitors with the ability to induce stomatal closure, including flg22, a yeast elicitor, and fungal elicitor (chitosan), is accompanied by ROS accumulation, NO synthesis, and (Ca 2+ ) cyt oscillations (11,12). These accompanying events are all common to ABA signaling. Thus, stomatal regulation by elicitors is suggested to share a common mechanism with ABA signaling. The results of the present study, which have revealed the involvement of MYB44, an ABA regulator, are consistent with this suggestion.
Stomatal closure is enhanced by root colonization by the rhizobacterium Bacillus subtilis FB17 (FB17) in order to restrict pathogen entry (14). ABA and SA pathways are considered to regulate the stomatal aperture in FB17-elicited ISR (14). This is also consistent with the finding of the involvement of MYB44, which activates ABA and WRKY70-mediated SA signals. Fig. 6 shows a summary of our results and the reported function of MYB44. GP17-2 induces disease resistance in plants partly via stomatal closure ( Fig. 1 and 2). The GP17-2induced suppression of stomatal reopening is mediated by the activation of MYB44 at the level of gene expression (Fig. 3  and 4). Activated MYB44 represses protein phosphatase 2Cs (PP2Cs) (ABI1, ABI2, AtPP2CA, HAB1, and HAB2) by the transcriptional suppression of PP2C genes through a repressor domain in MYB44 (9,23). This suppression of PP2Cs causes the activation of ABA signaling (9). Therefore, the stomatal aperture was smaller in MYB44 ox plants than in wild-type plants (Fig. 5), which is consistent with previous findings (9). We demonstrated that CF-enhanced MYB44 expression did not result in the closure of the stomata prior to the inoculation (Fig. 2), whereas MYB44 ox plants exhibited stomatal closure (Fig. 5). These results suggest that MYB44mediated stomatal closure is moderately regulated and will be enhanced by pathogen recognition. In contrast, COR produced by Pst has been shown to force stomatal reopening (20). Since the chemical structure of COR is similar to that of JA-Ile, COR has been suggested to activate JA signaling as an analog of JA-Ile (20). There are two possible mechanisms by which MYB44 suppresses stomatal reopening by COR. One is the antagonistic interaction of GP17-2-enhanced ABA signaling with pathogen-associated JA signaling (upper question Fig. 6. Model for effects of MYB44 in Penicillium simplicissimum GP17-2-elicited ISR. Penicillium simplicissimum GP17-2 (GP17-2) stimulates disease resistance in plants in part by stomatal closure (Fig. 1 and 2). Stomatal closure by GP17-2 is achieved by the enhancement of ABA signaling through the gene activation of MYB44 (Fig. 3 and 4). Activated MYB44 represses PP2Cs, leading to the activation of ABA signaling (9). Coronatine (COR) produced by Pseudomonas syringae pv. tomato DC3000 (Pst) forces stomatal reopening by inducing COI1-mediated JA signaling in plants (20). SnRKs, Snf1-related protein kinases. Open and closed circles indicate ion channels regulating the stomatal aperture. mark in the figure). Another is the regulation of ion channels (open and closed circles in the figure) in guard cells by GP17-2-enhanced ABA signaling (lower question mark in figure).
In conclusion, our study on GP17-2-elicited ISR suggest that the involvement of MYB44 in ISR and MYB44-mediated stomatal resistance is a common event in ISR.