Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.


Introduction
In nature, numerous potential pathogens such as fungi, bacteria, and viruses continually attack plants; however, disease development remains the exception. Plants are immune to most potential pathogens; this characteristic is referred to as a "nonhost resistance." Moreover, they have the ability to reduce the disease severity of actual pathogens. Plant innate immune responses to pathogens consist of a two-layer surveillance system that comprises pattern recognition receptors (PRRs) and intracellular nucleotide binding-leucine rich repeat (NLR) proteins, which are encoded by R (resistance) genes [1]. PRRs, which are localized on the surface of

HM-induced gene expression
Induced resistance is associated with the expression of disease resistance marker genes. To determine whether HM acts as an inducer of induced resistance in Arabidopsis thaliana, we investigated the expression profile of defense-related genes in response to HM treatment in A. thaliana Col-0 plants by quantitative real time-polymerase chain reaction (qRT-PCR). We used the A. thaliana PR-1 gene as a marker for the SA-dependent signal transduction pathway [11] and the PDF1.2 gene, which encodes a plant defensin, as a marker for the JA/ET pathway [12].
Expression of the PR-1 gene increased over time in Col-0 plants sprayed with 1250 ppm HM, with a peak at day 1, whereas the expression level continued to rise for 2 days in plants sprayed with 250 ppm HM (Fig. 1A). The expression of PDF1.2 initially increased with a peak at 10 h and 5 h in Col-0 plants sprayed with 1250 ppm and 250 ppm HM, respectively, which was earlier than that of the PR-1 gene. The timing of induction differed between the two representative marker genes. The expression of the PR-1 gene increased with time in Col-0 plants where the soil was drenched with 250 ppm HM (Fig. 1B). In addition, the expression of PR-1 and PDF1.2 increased with a peak at 2 days in Col-0 plants where the soil was drenched with 1250 ppm HM. The transcription levels of PR-1 and PDF1.2 were much higher in the Col-0 plants sprayed with HM than in plants where the soil was drenched.
To investigate the signal transduction pathways activated by HM, we examined other defense-related genes induced in response to treatment with HM using A. thaliana as a model system. Thus, we used qRT-PCR to analyze the effects of HM on the expression of the following A. thaliana putative marker genes: PR-2 (glucanase; BGL2), basic chitinase PR-3 (At3g12500), and chitinase (At2g43620; CHI620, At2g43570; CHI570). The expression levels of these genes were induced by 1250 ppm HM treatment (Fig. 1C). The expression levels of chitinase (CHI620, CHI570) and glucanase (PR-2), which are antipathogenic enzymes, also increased after HM treatment. The expression level of CHI620 increased early in Col-0 plants sprayed with HM. Thus, the plants recognized the HM components as MAMPs and increased the expression of defense-related genes.
HM protects Arabidopsis against Pseudomonas syringae pv. maculicola Induced resistance is characterized by the induction of disease resistance to various pathogens. Previously, induced resistance in A. thaliana has been shown to be effective against disease caused by P. syringae pv. tomato [13]. To determine whether HM acts as an inducer of induced resistance in A. thaliana Col-0, the plants were sprayed with water or HM 2 days prior to P. syringae pv. maculicola (Psm) inoculation. As shown in Fig. 2A, treatment with 1250 ppm HM decreased the disease symptoms caused by the pathogen. In contrast, water-treated plants inoculated with Psm exhibited chlorotic leaf spotting. In addition, bacterial growth was reduced in Col-0 plants sprayed with 1250 ppm HM. At 3 days postinoculation (dpi), the HM-treated plants contained 10-fold lower bacterial titers than the water-presprayed control leaves (Fig. 2B). Thus, reduced bacterial growth was responsible for the decrease in the disease symptoms. Furthermore, this concentration of HM did not cause any phytotoxicity. Moreover, bacterial growth decreased in Col-0 plants where the soil was drenched with 1250 ppm HM (Fig. 2C).
To determine whether HM treatment reduced bacterial growth in a dose-dependent manner, HM was applied at increasing concentrations and bacterial growth was measured (Fig. 2B). Compared with the control, HM concentrations as low as 250 ppm reduced bacterial growth in a dose-dependent manner.
We investigated whether the defense-related hormone SA plays a role in HM-induced activation of SAR using NahG transgenic Arabidopsis, which fails to accumulate SA [14]; eds16-1, which does not produce SA [15]; and npr1-1, which fails to activate PR gene expression [16]. Both JA and ET have been suggested to play important roles in plant defense against pathogen infection. To determine the roles of JA and ET in HM-induced resistance, we tested A. thaliana mutants that varied in their ability to respond to methyl jasmonate (jar1-1 is insensitive to JA [17]) or ET (ein2-12 is insensitive to ET [18]). HM was applied to these plants, and their resistance levels to Psm were determined. These mutants were treated with 1250 ppm HM at 2 days prior to bacterial inoculation. At 3 dpi, mutants deficient in the SA signaling pathways exhibited decreased induced resistance to Psm infection, whereas mutants deficient in the JA or ET signaling pathways were protected in a manner similar to the wild-type Col-0 (Fig. 3). The standard error (SE). The nucleotide sequences of the gene-specific primers for each gene are listed in Table 1. water-pretreated controls exhibited increased bacterial growth. Thus, HM appears to activate disease resistance to Psm via a pathway that is dependent on SA. In addition, HM has no direct toxic effect on bacteria.
We investigated whether HM potentiates defense responses during infection with pathogens. Treatment with HM potentiated Arabidopsis plants inoculated with Psm to induce expression of chitinase (CHI620) gene more intensively (Fig. 4). Thus, defense responses are boosted in HM-treated plants during the infection with Psm.
Furthermore, the expression of PR-1 and PDF1.2 genes in SA/ET/JA signaling-defective mutants were investigated for analyzing HM-primed defense responses after infection with Psm.
Accumulation of PR-1 transcripts in NahG transgenic plants, eds16-1 and npr1-1 mutants were lower than those in Col-WT, ein2-12 and jar1-1 mutants during the early phase of Figure 2. Effects of HM application on P. syringae pv. maculicola in Arabidopsis plants. The 35-day-old A. thaliana Col-0 plants were sprayed (A, B) or soil drenched (C) with water (control) or the indicated concentrations of HM at 2 days prior to spray inoculation with a bacterial suspension (10 8 cfu mL −1 ) of P. syringae pv. maculicola. Symptoms were observed 3 days after inoculation with the pathogen (A). Pathogen growth was determined 3 days after inoculation by assessing P. syringae pv. maculicola-rpoD mRNA by qRT-PCR (B, C). Bars indicate the standard error (SE). The asterisk indicates a significant difference compared with the control (Dunnett's method [35], P < 0.05). The experiment was repeated at least twice with similar results.  . Induction of resistance to P. syringae pv. maculicola by HM application in Arabidopsis defense signaling-defective mutants. The 35-day-old A. thaliana mutants were sprayed with water (control) or 1250 ppm HM at 2 days prior to spray inoculation with a bacterial suspension (10 8 cfu mL −1 ) of P. syringae pv. maculicola. Pathogen growth was determined 3 days after inoculation by assessing P. syringae pv. maculicola-rpoD mRNA by qRT-PCR. Bars indicate the standard error (SE). The asterisk indicates a significant difference compared with the control (Dunnett's method [35], P < 0.05). The experiment was repeated at least three times with similar results.  thaliana Col-0 plants were sprayed with water (control) or 1250 ppm HM at 2 days prior to spray inoculation with a bacterial suspension (10 8 cfu mL −1 ) of P. syringae pv. maculicola. Aboveground tissues were collected 10, 24, 72 h after inoculation. Total RNA was extracted, and first-strand cDNA was synthesized for expression analysis. Expression levels of the chitinase gene (At2g43620; CHI620) were monitored by qRT-PCR. The expression level of each gene was normalized against the expression level of CBP20, which is constitutively expressed. Each experiment was repeated at least twice with similar results. Bars indicate the standard error (SE). The nucleotide sequences of the genespecific primers for each gene are listed in Table 1.  . HM-primed defense responses after the infection with P. syringae pv. maculicola in Arabidopsis defense signaling-defective mutants. The 35-day-old A. thaliana Col-0 plants and defense signaling-defective mutants were sprayed with water (control) or 1250 ppm HM at 2 days prior to spray inoculation with a bacterial suspension (10 8 cfu mL −1 ) of P. syringae pv. maculicola. Aboveground tissues were collected 10 h after inoculation. Total RNA was extracted, and first-strand cDNA was synthesized for expression analysis. Expression levels of the PR-1 and PDF1.2 genes were monitored by qRT-PCR. The expression level of each gene was normalized against the expression level of CBP20, which is constitutively expressed and is shown as the reference value. Each experiment was repeated at least twice with similar results. Bars indicate the standard error (SE). Means labeled with the same letters are not statistically different at the 5% confidence level based on Tukey's test. The nucleotide sequences of the gene-specific primers for each gene are listed in Table 1  Does HM activate chitin signaling?
Chitin is a representative MAMP molecule for various fungi, and its perception by PRRs triggers various plant defense responses [19,20]. Chitin elicitor receptor kinase 1 (CERK1) is an essential molecule for chitin perception and chitin elicitor signaling in A. thaliana [21]. In the present study, compared with control plants, an HM-treated cerk1 mutant exhibited increased resistance to Psm (Fig. 3).

HM-induced disease resistance to Colletotrichum higginsianum
To test whether HM acts as an inducer of resistance to fungal pathogens, A. thaliana plants were sprayed with water or HM at 2 days prior to C. higginsianum inoculation. The results showed that the infection process was not completely stopped by HM treatment; however, the disease incidence was moderately reduced (Fig. 6A). In contrast, no enhanced resistance to C. higginsianum was detected in the Col-0 plants grown in soil drenched with 1250 ppm HM (Fig. 6B). HM was applied to transgenic plants and mutants, and their resistance to C. higginsianum was evaluated (Fig. 7). Enhanced resistance to C. higginsianum was not detected in NahG transgenic plants and eds16-1 and npr1-1 mutants that had been pretreated with 1250 ppm HM at 2 days prior to fungal inoculation. In contrast, HM-treated ein2-12 and jar1-1 mutants did not support the fungal growth, whereas water-pretreated controls exhibited increased fungal growth.
Thus, HM appears to activate disease resistance to C. higginsianum via an SA-dependent pathway. Compared with control plants, the HM-treated cerk1 mutant also exhibited increased resistance to C. higginsianum (Fig. 7). In addition, HM exhibited no direct toxic effect on fungi.
HM protects Brassica crops against P. cannabina pv. alisalensis P. syringae pv. alisalensis was recently renamed as P. cannabina pv. alisalensis (Pca) [22]. To test whether HM induces resistance in Brassica rapa var. chinensis, plants treated with HM were inoculated by spraying with Pca. The results revealed that HM protects Brassica crops against Pca (Fig. 8). Thus, HM appears to activate disease resistance in Brassica crops.
Discussion YCWE has not been tested in practice as an agricultural chemical. However, it may act as MAMP and lead to induced resistance in plants. The mode of action of YCWE is not well known in plants; therefore, we analyzed the protective effect of this product against bacterial and fungal pathogens in A. thaliana and Brassica plants. In A. thaliana and Brassica plants, we observed that HM prepared from YCWE induced the expression of plant defense-related genes and resistance to Psm and Pca, which are similar pathogens that are combated via SA-induced resistance. The infection process of the hemibiotrophic fungal pathogen C. higginsianum was not completely stopped by HM treatment; however, plants treated with HM were moderately protected against the pathogen. Thus, HM protects different Brassicaceae species against bacterial and fungal pathogens, demonstrating the broad range of activity of this product. Importantly, HM has no direct toxic effect on bacteria or fungi. HM-mediated resistance is probably based on the activation of host resistance mechanisms.
Yeast cell walls comprise polysaccharides, i.e., approximately 40% of mannoproteins, approximately 60% of β-glucan, and approximately 2% of chitin [9,23], whereas HM contains approximately 1.3% of chitin. Chitin-induced defense responses were completely suppressed in the cerk1 mutant [21]; thus, the present study indicated that HM-induced resistance involved chitin-independent disease resistance to bacterial and fungal pathogens in A. thaliana.
The NahG transgenic plants and eds16-1 and npr1-1 mutants exhibited reduced induced resistance to Psm and C. higginsianum after HM treatment, indicating that SA synthesis and NPR1 play a role in HM-elicited defense responses of A. thaliana against Psm and C. higginsianum. In addition, the results demonstrated that HM does not exhibit a direct antibiotic effect on Psm and C. higginsianum. The impaired synthesis of SA, EDS16, and NPR1 in defense mechanisms may be disadvantageous for plants. In contrast, the defective mutants of JA and ET signaling pathways, jar1-1 and ein2-12, respectively, were protected against Psm and C. higginsianum with an effect resembling the wild-type Col-0 control. The plant hormones JA and ET have been implicated in an alternative defense transduction pathway that is separate from SA [24]. HM-induced resistance to these pathogens was not dependent on the sensitivity to JA and ET, which excludes the involvement of JA-and ET-mediated defense signaling mechanisms. A previous study revealed that β-aminobutyric acid (BABA), which is known to be a disease resistance inducer, did not protect transgenic Arabidopsis NahG plants and npr1-1 Figure 6. Effect of HM application on Colletotrichum higginsianum in Arabidopsis plants. The 28-to 30-day-old A. thaliana Col-0 plants were sprayed (A) or soil drenched (B) with water (control) or the indicated concentrations of HM at 2 days prior to spray inoculation with a spore suspension (5 × 10 5 spores mL −1 ) of C. higginsianum. Pathogen growth was determined 5 days after inoculation by assessing C. higginsianum actin mRNA by qRT-PCR. Bars indicate the standard error (SE). The asterisk indicates a significant difference compared with the control (Dunnett's method [35], P < 0.05). The experiment was repeated at least twice with similar results. doi:10.1371/journal.pone.0115864.g006 mutants against P. syringae pv. tomato DC3000 (Pst DC3000) [25]. Lawton et al. revealed that benzothiadiazole (BTH)-treated A. thaliana plants exhibited resistance to Pst DC3000 [26]. In addition, BTH treatment induced disease resistance in NahG transgenic A. thaliana plants, suggesting that the action of BTH does not require SA accumulation [26]. However, in contrast to BTH, HM and BABA require SA accumulation and enhance resistance via plant defense responses.
The protective action of HM against Psm and C. higginsianum seems to act as a disease resistance inducer or a plant defense activator but not as a bactericide and fungicide. Therefore, we analyzed the expression of plant defense-related genes induced by HM treatment. The results indicated that HM treatment elicited the induction of several defense-signaling pathways, including SA, JA, and ET signaling. There is antagonistic signaling cross-talk between the Figure 7. Induction of resistance to C. higginsianum by HM application in Arabidopsis defense signaling-defective mutants. The 28-to 30-day-old A. thaliana mutants were sprayed with water (control) or 1250 ppm HM at 2 days prior to spray inoculation with a spore suspension (5 × 10 5 spores mL −1 ) of C. higginsianum. Pathogen growth was determined 5 days after inoculation by assessing C. higginsianum actin mRNA by qRT-PCR. Bars indicate the standard error (SE). The asterisk indicates a significant difference compared with the control (Dunnett's method [35], P < 0.05). The experiment was repeated at least twice with similar results.
doi:10.1371/journal.pone.0115864.g007 Figure 8. HM application induced resistance against P. cannabina pv. alisalensis in Brassica rapa var. chinensis. Brassica rapa var. chinensis seeds were sown in soil mixed with 5 g L −1 of raw HM material powder. Two-week-old seedlings were inoculated at approximately the one true leaf stage by spraying with a bacterial suspension (10 6 cfu mL −1 ) of P. cannabina pv. alisalensis. Observations of bacterial leaf spot symptoms were made at 7 dpi. The percentage of infected leaves (IL) was calculated as follows: IL = (number of leaves with any chlorotic leaf spots)/(total number of leaves), Data represent mean ± SE (Trial 1: n = 12, Trial 2: n = 23). Asterisks indicate significant differences compared with the controls (Welch's t-test [36]).
doi:10.1371/journal.pone.0115864.g008 defense signal transduction pathways in A. thaliana and tobacco [27][28][29][30]. The maximum induction timing for two representative marker genes of plant defense responses, PR-1 and PDF1.2, differed in A. thaliana treated with HM. Thus, HM treatment appeared to induce early activation of the JA/ET signaling pathway and late activation of the SA signaling pathway. The timing of activation of these signaling pathways may be essential for plant resistance to pathogenic infections. Therefore, the activation of SA and JA/ET defense signaling pathways at different times and our analyses using signaling mutants of these pathways suggest that HM could control various diseases.
HM-primed and -induced defense responses after the infection with Psm were investigated by analyzing the expression of defense-related genes in defense signaling-defective mutants and wild-type plants. The expression of PR-1 gene in response to Psm was reduced in HMtreated SA signaling-defective mutants indicating susceptible to Psm. On the contrary, PR-1 transcripts were potentiated in ET/JA signaling-defective mutants indicating resistance to Psm. As a consequence, SA-mediated defense responses are involved in resistance to Psm.
In addition, expression of CHI620 gene was strongly induced in resistance interaction of HM-treated plants with Psm, but weekly induced in susceptible interaction. Therefore, HM treatment prime and boost defense responses leading to restriction of Psm growth.
Our results indicate that HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, HM facilitated induced resistance to pathogens on plant leaves, which enhanced the expression of plant defense-related genes, i.e., PR gene expression. Our experiments suggest that HM could be a useful tool in the effective control of plant disease.

Preparation of Housaku Monogatari (HM)
HM was produced using a by-product, the yeast cell wall extract (YCWE), prepared from the budding yeast Saccharomyces pastorianus during the beer brewing process. The yeast slurry collected after beer brewing was treated with protease YL-15 (Amano Enzyme Inc., Nagoya, Japan). YCWE was obtained by removing the supernatant following centrifugation. To digest YCWE to a moderate molecular size, YCWE was treated again with 0.5% YL-15 at 55°C for 18 h and powdered HM was produced by drum drying. Similar to general yeast cell wall composition [9], HM contains polysaccharides (15%-25% of β-glucan, 5%-15% of α-glucan, 10%-20% of mannan, and 0.5%-2% of chitin).

Plant materials
Arabidopsis thaliana accession Columbia-0 (Col-0) was obtained from RIKEN BRC (Tsukuba, Japan). Before inoculation, the plants were grown in Soil-mix (Sakata Seed Corp., Yokohama, Japan) and expanded vermiculite (1.5-2 mm granules) at a ratio of 1:2 for 28-35 days in a growth chamber at 22°C with 12-h light.

HM treatment
A. thaliana plants were treated with water (control) or the indicated concentration of HM, containing a spreader (0.01%) for spraying but not for soil drenching.

Expression analysis of defense-related genes in Arabidopsis plants
The expression levels of the PR-1 and PDF1.2 genes were monitored by quantitative real timepolymerase chain reaction (qRT-PCR), as described previously [31]. PR-2 (glucanase; BGL2), basic chitinase PR-3, and chitinase (At2g43620; CHI620, At2g43570; CHI570) gene expression levels were also monitored by qRT-PCR. The primers used for qRT-PCR are listed in Table 1.
Pseudomonas syringae pv. maculicola infection and quantification of rpoD mRNA Spontaneous rifampicin-resistant colonies of P. syringae pv. maculicola (MAFF302783Rif4) (Psm) were obtained by culturing the strain MAFF302783 in King's B medium containing 100 μg mL −1 of rifampicin. Psm was grown in liquid King's B medium containing rifampicin (25 μg mL −1 ). Bacteria were harvested by centrifugation, and the cell pellets were washed with 10 mM MgSO 4 before resuspension in 10 mM MgSO 4 at a concentration of 1 × 10 8 cfu mL −1 for in planta growth assays. Five-week-old Arabidopsis Col-0 plants (susceptible to Psm) and the mutants were used in virulence assays. The plants were inoculated by spraying the leaves with the bacterial suspension. The inoculated plants were then placed in a growth chamber with 100% relative humidity at 22°C (12-h light cycle). Bacterial growth was determined 3 days after inoculation, and pathogen growth was determined by measuring the rpoD mRNA level by qRT-PCR, as described previously [32].
Plants inoculated with C. higginsianum were harvested at 5 days postinoculation (dpi) for qRT-PCR, and C. higginsianum was quantified as described previously [34]. P. cannabina pv. alisalensis infection B. rapa var. chinensis seeds were sown in soil mixed with 5 g L −1 of HM raw material powder. Two-week-old seedlings at approximately the one true leaf stage were inoculated by spraying with a bacterial suspension (10 6 cfu mL −1 ) of P. cannabina pv. alisalensis. Observations of bacterial leaf spot symptoms were made at 7 dpi. The percentage of infected leaves (IL) was calculated as follows: IL = (number of leaves with any chlorotic leaf spots)/(number of leaves)