Insights on the Impact of Arbuscular Mycorrhizal Symbiosis on Eucalyptus grandis Tolerance to Drought Stress

Arbuscular mycorrhizal (AM) fungi play an important role in improving plant growth and development under drought stress. The MAPK cascade may regulate many physiological and biochemical processes in plants in response to drought stress. ABSTRACT Drought stress has a negative impact on plant growth and production. Arbuscular mycorrhizal (AM) fungi, which establish symbioses with most terrestrial vascular plant species, play important roles in improving host plant mineral nutrient acquisition and resistance to drought. However, the physiological and molecular regulation mechanisms occurring in mycorrhizal Eucalyptus grandis coping with drought stress remain unclear. Here, we studied the physiological changes and mitogen-activated protein kinase (MAPK) cascade gene expression profiles of E. grandis associated with AM fungi under drought stress. The results showed that colonization by AM fungi significantly enhanced plant growth, with higher plant biomass, shoot height, root length, and relative water content (RWC) under drought conditions. Mycorrhizal plants had lower levels of accumulation of proline, malondialdehyde (MDA), H2O2, and O2·− than seedlings not colonized with AM fungi. In addition, mycorrhizal E. grandis also had higher peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities under drought conditions, improving the antioxidant system response. Eighteen MAPK cascade genes were isolated from E. grandis, and the expression levels of the MAPK cascade genes were positively induced by symbiosis with AM fungi, which was correlated with changes in the proline, MDA, H2O2, and O2·− contents and POD, SOD, and CAT activities. In summary, our results showed that AM symbiosis enhances E. grandis drought tolerance by regulating plant antioxidation abilities and MAPK cascade gene expression. IMPORTANCE Arbuscular mycorrhizal (AM) fungi play an important role in improving plant growth and development under drought stress. The MAPK cascade may regulate many physiological and biochemical processes in plants in response to drought stress. Previous studies have shown that there is a complex regulatory network between the plant MAPK cascade and drought stress. However, the relationship between the E. grandis MAPK cascade and AM symbiosis in coping with drought remains to be investigated. Our results suggest that AM fungi could improve plant drought tolerance mainly by improving the antioxidant ability to protect plants from reactive oxygen species (ROS) and alleviate oxidative stress damage. The expression of the MAPK cascade genes was induced in mycorrhizal E. grandis seedlings under drought stress. This study revealed that MAPK cascade regulation is of special significance for improving the drought tolerance of E. grandis. This study provides a reference for improving mycorrhizal seedling cultivation under stress.

by regulating plant physiological and molecular responses and that the expression levels of MAPK cascade genes in E. grandis are influenced by AM symbiosis.
Here, we use a multidisciplinary approach that focused on antioxidant systems, osmotic adjustment, and the expression of E. grandis MAPK cascade genes. We characterized the E. grandis response to drought stress during the AM symbiosis process. In conclusion, our results showed that AM symbiosis positively affected drought tolerance in E. grandis by regulating physiological and molecular parameters.

RESULTS
Arbuscular mycorrhizal colonization promotes E. grandis tolerance to drought stress. E. grandis seedlings colonized with Rhizophagus irregularis (AM) formed typical mycorrhizal structures under well water (WW), middle drought (MD), and extreme drought (ED) conditions. However, no mycorrhizal colonization was observed in nonmycorrhizal (NM) E. grandis. AM fungi and E. grandis established a symbiotic relationship, and numerous hyphae and typical arbuscular structures could be observed in the epidermal and cortical cells of the mycorrhizal E. grandis seedling roots (Fig. 1A). While there were no significant differences in the total percent colonization frequency (F%) and mycorrhization intensity (M%) during the different water treatments ( Fig. 1B and  C), the arbuscular abundance in E. grandis roots under WW conditions was higher than that under ED conditions (Fig. 1D). AM E. grandis grew better than the NM plants, and drought stress made E. grandis shorter, with etiolated leaves ( Fig. 2A to C). The relative water content (RWC) is usually considered an important factor that reflects the water physiological conditions of plant tissues. Under WW and MD conditions, there were no significant differences in the RWC of plant leaves between the AM and NM plants, while the RWC of AM plants was significantly higher than that of the NM plants under ED stress (Fig. 2D). A comparison of AM and NM E. grandis revealed significant differences in E. grandis growth, including the plant fresh weight of shoots and roots, shoot height, and root length. The biomass, shoot height, and root length were significantly decreased in NM E. grandis seedlings compared with those in AM plants under MD and ED conditions (Fig. 2E to H). FIG 1 Arbuscular mycorrhizal colonization in E. grandis inoculated with R. irregularis under well water (WW), middle drought (MD), and extreme drought (ED) conditions. (A) Fluorescence microscopy images of R. irregularis-colonized roots of E. grandis after WGA488 staining under WW, MD, and ED conditions. a, arbuscule; in, internal hypha. Bars, 100 mm. (B to D) Total mycorrhizal frequency (B), mycorrhizal intensity (C), and arbuscule abundance (D) in R. irregularis-colonized roots estimated after WGA488 staining. The data are shown as the means 6 SE from three biological replicates (n = 3). Different letters indicate significant differences at a P value of ,0.05, according to one-way ANOVA and Tukey's test.
Effects of AM symbiosis on the contents of proline and other antioxidant substances (MDA, H 2 O 2 , and O 2 ·2 ) in E. grandis in response to drought. Reactive oxygen species (ROS) are inevitably induced when plants face environmental stress and have toxic effects on plants. To examine drought stress-induced ROS production in E. grandis, we assessed the proline, malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), and superoxide anion radical (O 2 Á2 ) contents of E. grandis seedling shoots and roots. Under WW conditions, the proline, MDA, H 2 O 2 , and O 2 Á2 contents in E. grandis seedling shoots and roots remained at low levels under both AM and NM treatments, and they were not significantly different. Under drought conditions, the proline content in shoots or roots increased significantly, while the AM plants showed lower proline contents than the NM plants (Fig. 3A). AM colonization significantly reduced the MDA contents in E. grandis shoots and roots compared with the NM plants in response to drought (Fig. 3B). The H 2 O 2 and O 2 Á2 accumulation patterns were similar to the proline and MDA accumulation patterns in E. grandis, with decreased H 2 O 2 and O 2 Á2 contents in mycorrhizal E. grandis compared to nonmycorrhizal E. grandis ( Fig. 3C and D). These data suggest that AM fungal colonization regulates E. grandis antioxidant substances during drought stress.
Response of antioxidant enzyme activity to drought stress in mycorrhizal and nonmycorrhizal E. grandis. To investigate the effect of drought stress on the antioxidant system during AM symbiosis, we determined the contents of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), which can reflect the activation levels of plant antioxidant enzymes. Under WW conditions, the POD activity in the shoots or roots of mycorrhizal E. grandis seedlings was increased, but it was not significantly different from that under NM conditions, while under MD and ED conditions, the POD activities in NM and AM seedlings were significantly different. The POD activity was much higher in AM plants than in NM E. grandis ( Fig. 4A and D). Similar expression patterns were observed for the SOD and CAT activities in AM and NM E. grandis plants. Drought stress induced significant increases in the SOD and CAT activities in plant roots and shoots, and mycorrhizal E. grandis had higher activities than nonmycorrhizal E. grandis (Fig. 4B, C, E, and F). These results showed that AM fungus colonization had positive effects on POD, SOD, and CAT activities under drought stress.
Relative expression levels of drought resistance genes in E. grandis and R. irregularis. To further investigate the molecular mechanisms of AM fungus symbiosis in response to drought, we analyzed the expression levels of drought resistance genes in E. grandis and R. irregularis using quantitative real-time PCR (qRT-PCR) technology. According to the released genome of E. grandis (48), plasma-membrane-intrinsic proteins (EgPIP1 and EgPIP2) and tonoplast-intrinsic proteins (EgTIP1 and EgTIP2) were identified using related PIP and TIP genes in other plants (28,29). We also identified aquaporin (RiAQP1, -2, and -3), trehalose-6-phosphate synthase (TPS) (RiTPS1 and -2), neutral trehalase (NTH) (RiNTH1), and Ri14-3-3 genes in R. irregularis according to a previous study (52). There were no significant differences in the expression levels between AM and NM plants under WW conditions. However, under drought stress, the expression levels of EgTIP1, EgTIP2, EgPIP1, and EgPIP2 were increased significantly, and the expression levels in the AM plants were highly upregulated compared with the NM plants ( Fig. 5A to D). RiAQP2, RiTPS2, RiNTH1, and Ri14-3-3 were expressed at high levels in response to MD  MAPK proteins from E. grandis are conserved across plant species. We initiated a search for the MAPK cascade genes in E. grandis using the related MAPK cascade genes in A. thaliana as reference sequences (53). The related MAPK cascade genes from E. grandis were identified by BLAST searches according to the released genome of E. grandis (48). To investigate the evolutionary relationships of MAPK cascade genes in E. grandis, we performed a phylogenetic analysis to construct a phylogenetic tree, which included 39 MAPK protein sequences from E. grandis and A. thaliana (Fig. 6). Studies on MAPK genes in A. thaliana have contributed significantly to research on MAPK cascades in other plants; therefore, we chose A. thaliana as a reference to analyze its homology with E. grandis. The E. grandis MAPK proteins were divided into three groups: MAPKKK, MAPKK, and MAPK. The EgMPKKK-1, EgMPKKK-2, and EgMPKKK-3 proteins are closely related to MAPKKK proteins of A. thaliana. Moreover, the EgMKK1, 3, 6, 5-1, 5-2, 9-1 and 9-2 proteins from E. grandis belong to the MAPKK group, and the EgMPK1, 6-1, 6-2, 7, 9-1, 9-2, -1, and -2 had high similarity to the MAPK group in A. thaliana (Fig. 6A). This result indicated the conserved evolutionary origin of the FIG 5 Relative expression levels of the stress response genes EgTIP1, EgTIP2, EgPIP1, and EgPIP2 from E. grandis (A to D) and RiAQP1, -2, and -3; RiTPS1 and -2; RiNTH1; and Ri14-3-3 from R. irregularis in the mycorrhizal roots of E. grandis seedlings (D to K) under different drought stress conditions. Letters indicate significant differences between treatments at a P value of ,0.05, based on one-way ANOVA and Tukey's tests. Error bars represent data from three biological replicates with SE values.
AM Symbiosis Benefits E. grandis Response to Drought Microbiology Spectrum MAPK proteins in E. grandis. We also identified common motifs of E. grandis MAPK proteins using the Multiple Expectation Maximization for Motif Elicitation (MEME) website (http:// meme-suite.org/tools/meme). In the group of MAPKKKs, EgMPKKK-2 and EgMPKKK-3 contained four motifs, while EgMPKKK-1 contained five motifs. Most members of the MAPKK group contained five motifs, except for EgMKK1, which contained four motifs.
In the MAPK group, EgMPK1, EgMPK9-1, and EgMPK9-2 contained eight motifs, and the other members contained nine motifs (Fig. 6B). These results showed that the MAPK pathway of E. grandis may have the biological functions similar to those of the MAPK proteins of A. thaliana. Expression patterns of E. grandis MAPK cascade genes during AM symbiosis in response to drought stress. In order to investigate the function of E. grandis MAPK cascade genes during AM symbiosis in response to drought, we analyzed the expression profiles of the MAPK cascade genes of E. grandis under WW, MD, and ED conditions. For all of the MAPK cascade genes, no significant changes were observed between AM and NM plants under WW treatment. However, the expression levels of these MAPK genes increased significantly under both MD and ED treatments (Fig. 7). Accordingly, the transcript levels of EgMPKKK-1, EgMKK9-1, EgMPK1, EgMPK7, and EgMPK-1 were significantly upregulated in mycorrhizal roots under ED conditions (Fig. 7A, I, K, N, and Q), while under both MD and ED conditions, the expression levels of EgMPKKK-2, EgMPKKK-3, EgMKK1, EgMKK6, EgMKK5-1, EgMKK5-2, EgMKK9-2, EgMPK6-1, EgMPK6-2, EgMPK9-1, EgMPK9-2, and EgMPK-2 were obviously upregulated in AM E. grandis compared with the control plants (NM) (Fig. 7). In conclusion, these results indicated that AM fungus symbiosis affected the expression of MAPK cascade genes in E. grandis in response to drought stress.
Correlation of the regulation of E. grandis MAPK cascade genes and physiological parameters. For the purpose of demonstrating the relationship between the expression levels of E. grandis MAPK cascade genes and the antioxidant system, we performed redundancy analysis (RDA) using RStudio. The results showed that the POD, SOD, CAT, proline, MDA, H 2 O 2 , and O 2 Á2 physiological indicators in plant roots or shoots had acute angles with the first sorting axis, RDA1, and the arrows of them pointed toward the drought region, indicating that all of these indicators change in response to drought (Fig. 8). In addition, we found that the arrows of MAPK cascade genes were also distributed mainly in the drought region. EgMPK9-1, EgMPK-2, EgMKK6, EgMPK6-1, EgMPKKK-2, EgMPKKK-3, EgMKK9-2, and EgMKK1 were the main genes that affected the antioxidant system of plant roots (Fig. 8A), while EgMPK9-1, EgMPK-2, EgMKK1, EgMKK6, EgMKK5-1, EgMPK6-1, EgMPKKK-2, EgMPKKK-3, EgMKK9-2, and EgMPK1 mainly maintained the osmotic balance of plant leaves in response to drought (Fig. 8B), and EgMPK9-1, EgMPK-2, EgMKK6, and EgMKK1 may be more likely to be expressed in mycorrhizal E. grandis under drought conditions in plant leaves and roots. EgMPK9-1, EgMPK-2, and EgMPKKK-3 may be the main genes affecting the antioxidant system of E. grandis by improving drought resistance because they had the longest projection lengths on the first RDA1 axis in both leaves and roots (Fig. 8).

DISCUSSION
Although AM fungi improve the drought resistance of plants, which has been confirmed in diverse plants species (5,54,55), very few of them have been researched in E. grandis trees; especially, the molecular features of MAPK cascade proteins in this tree species are not well known. In our research, E. grandis seedlings were colonized with R. irregularis to establish a symbiotic relationship. We demonstrate that AM symbiosis improves the drought tolerance of E. grandis by regulating the antioxidant system and upregulating the expression of some MAPK cascade genes.
AM symbiosis improves physiological activity in E. grandis under drought stress. In our research, the AM fungus R. irregularis and E. grandis seedlings establish a symbiotic relationship. We found that the mycorrhizal colonization level in E. grandis was not influenced by different water conditions (Fig. 1). This result is in agreement with the results of previous studies (31,56,57). We suspected that this was because drought treatment was performed at 42 days of colonization with AM fungi, which is not sufficient to influence the AM fungus colonization rate. Mycorrhizal E. grandis showed better performance, with increased fresh weight, plant height, and root length, under well water and drought conditions (Fig. 2). This might be the reason why AM fungi in the roots of E. grandis seedlings contribute to the formation of hyphal structures connecting with the soil. These hyphae can penetrate the soil and expand the root absorption area. Therefore, AM fungi can help plants acquire more water and nutrients to retain normal physiological functions in response to stressful conditions. Similar results were also described previously for other plant species (58,59). Under ED conditions, the RWC of leaves in AM plants was increased compared with that in NM plants, which also showed that AM symbiosis helps plants absorb water.
Under stress conditions, ROS accumulate in large quantities in plants (60). Usually, plants initiate the antioxidant system to protect themselves from ROS accumulation when facing environmental stress conditions (61,62). Antioxidant enzymes, including POD, SOD, and CAT, are considered important factors in reducing ROS accumulation (63,64). In our experiments, the activities of POD, SOD, and CAT were significantly higher in E. grandis colonized with AM fungi than in nonmycorrhizal plants (Fig. 4). This trend was consistent with the patterns in the contents of active substances (MDA, H 2 O 2 , and O 2 Á2 ) under drought stress. The contents of MDA, H 2 O 2 , and O 2 Á2 were increased significantly in E. grandis seedlings, but their contents were lower in AM plants than in NM plants (Fig. 3). Water-stressed plants have been reported to accumulate proline to enhance the ability for resistance to drought (56,65). We also verify that the proline content was higher in NM plants than in AM plants under drought stress conditions. A lower proline content suggests improved tolerance to drought (66,67). Therefore, AM symbiosis maintains ROS accumulation in E. grandis seedlings at a relatively stable level by increasing antioxidant abilities under drought stress, thereby reducing damage to cells and alleviating serious damage caused by drought stress. Drought resistance genes of AM fungi and E. grandis during the regulation of mycorrhizal symbiosis in response to drought stress. AM symbiosis can alleviate the negative effects of drought stress by regulating the expression of drought resistance genes such as the aquaporin-related genes. Previous studies have shown that AQP genes in Glomus intraradices transport water to host plants, and the expression levels of GintAQPF1 and GintAQPF2 were higher under water stress (30,68). Trehalose-6-phosphate synthase (TPS) genes can improve resistance to drought in various plants and fungi (69). Neutral trehalase (NTH) was also an important factor during drought (70). The Ri14-3-3 protein in R. irregularis is essential for arbuscule formation (71). We also analyzed the expression levels of RiAQP1, RiAQP2, RiTPS1, RiTPS2, RiNTH1, and Ri14-3-3 from R. irregularis in mycorrhizal E. grandis, which were identified previously by Wang et al. (52). We observed that the transcriptional levels of RiAQP1, RiAQP2, RiTPS1, RiTPS2, RiNTH1, and Ri14-3-3 were increased significantly under ED conditions (Fig. 6E to K). Meanwhile, some studies also showed that AM symbiosis affected the expression of some PIP and TIP genes in plants (28,29). Similar results were observed in our research, as increased expression levels of EgPIP1, EgPIP2, EgTIP1, and EgTIP2 were found in mycorrhizal plants in response to MD and ED conditions (Fig. 6A to D). All of these results imply that AM fungus symbiosis may enhance plant drought tolerance by increasing the expression of drought resistance genes. The higher expression levels of all of these genes might lead to a better water uptake environment.
The MAPK cascade regulates the physiological responses of mycorrhizal E. grandis to drought. When plants are facing drought stress, they receive and transduce drought signals to regulate physiological and molecular responses to adapt to external stress. Fortunately, the MAPK signal transduction cascade is considered to have a critical role in the processes of regulating the immune responses to drought (53,72,73). Previous studies have reported that during AM fungus colonization of plant roots, the expression levels of MAPK cascade genes in AM fungi and plant roots increased significantly in response to drought (45,47). In our study, 18 MAPK cascade genes were isolated from E. grandis, and they are conserved based on the phylogenetic relationships between A. thaliana and E. grandis, the motifs of which also showed evolutionary conservation in E. grandis (Fig. 5). The expression levels of related MAPK cascade genes in E. grandis were significantly upregulated in mycorrhizal plant roots under drought conditions (Fig. 7). AM symbiosis also has an impact on the expression levels of some MAPK cascade genes in other plant species such as Populus simonii Â P. nigra (27), soybean (45), and Malus hupehensis (47). The MAPK cascade, as an essential regulator of antioxidant defense, can alter the expression profiles of antioxidant systems under diverse stress conditions (74). Previous studies reported that MAPK cascade genes directly regulate the antioxidant system by interactions with ROS (75). In addition, the expression of CAT1 is activated by MPK6 operating downstream of MKK1 during salt stress or after drought and ABA treatments (39,76). Our results also showed that there is a high correlation between the expression of MAPK cascade genes and physiological parameters in E. grandis under drought stress. Antioxidant enzymes (POD, SOD, and CAT) may be related to the expression of MAPK cascade genes (EgMPK6-1 and EgMKK5-1) in mycorrhizal E. grandis under drought conditions. Our research revealed that AM fungus symbiosis has a positive influence on the regulation of MAPK cascade genes in E. grandis. The MAPK cascade genes in mycorrhizal E. grandis seedlings may respond to drought stress by enhancing the antioxidant system and osmotic regulation. However, the potential functions and regulation of metabolism through these intricate interactions of MAPK cascade proteins remain unclear, for which further study is necessary.
In summary, we suggest that AM symbiosis alleviates the negative effects of drought stress on E. grandis seedlings by changing the plant's physiology and MAPK cascade gene expression. Under drought stress, the expression levels of drought resistance genes from E. grandis and AM fungi were elevated significantly. The AM fungus R. irregularis improves the drought resistance of E. grandis by altering osmotic regulation, the antioxidant system, and the expression of MAPK cascade genes. Our study provides physiological and molecular evidence for the effects of AM fungus colonization on improving drought resistance and provides insights into the expression of MAPK cascade genes in E. grandis, which will provide new thoughts for improving mycorrhizal seedling cultivation under stress.

MATERIALS AND METHODS
Plant material, AM fungus colonization, and growth conditions. Eucalyptus grandis seeds were provided by the Research Institute of Tropical Forestry (China Academy of Forestry, Guangzhou, China). E. grandis seeds were surface sterilized in 3% sodium hypochlorite for 20 min and then washed with sterile distilled water three times. The seeds were germinated in 1/4-strength Murashige-Skoog basal salt mixture agar medium at 25°C in the dark for 3 days, and the seedlings were then transferred to a growth chamber programmed for 16 h of light at 26°C and 8 h of darkness at 20°C for 14 days. After 14 days of cultivation, the seedlings were cultivated in small plastic pots (8 by 8 by 8 cm) with sterilized quartz sand. The roots of E. grandis seedlings were colonized with about 500 spores/plant.
The AM fungus used in this study is R. irregularis DAOM197198, which was purchased from Agronutrition, Toulouse, France.
Drought treatments. E. grandis plants grown in pot cultures were inoculated with (AM) or without (NM) R. irregularis. In the early stages of growth, all E. grandis seedlings were watered with a modified Long-Ashton (mLA) nutrient solution twice a week (77). After 42 days of inoculation, three kinds of water conditions were established in the experiments: WW (well water), with 75% field water capacity (FWC); MD (middle drought), with 50% FWC; and ED (extreme drought), with 25% FWC. The FWC was measured as described previously (78). There were 3 replicates per treatment. The soil moisture content was maintained by weighing at a fixed time every day. The drought treatment lasted for an additional 14 days; next, some mycorrhizal E. grandis root samples were used for colonization analysis, and the remaining root and shoot tissues were immediately frozen in liquid nitrogen and stored at 280°C for subsequent analyses.
Quantification of mycorrhizal colonization. Fresh roots of mycorrhizal E. grandis were immersed in 10% KOH at 90°C for 10 h, changing to a fresh KOH solution every 2 h. The roots were then neutralized in 2% HCl for 10 min and washed three times with sterile water. The samples were then stained with 5.0 mg/mL wheat germ agglutinin (WGA)-Alexa Fluor 488 conjugates (WGA488; Invitrogen, USA) for 30 min at 37°C. The roots were washed in 1Â Hanks' balanced salt solution without calcium, magnesium, and phenol red. The mycorrhizal levels of E. grandis were determined by WGA488 staining, and colonization was quantified using the MYCOCALC program as described previously (79). AM fungal structures were examined and captured using a confocal microscope (Y-TV55; Nikon).
Plant relative water content, biomass, height, and root length. When the E. grandis seedlings were harvested, the above-ground and underground weights, plant height, and root length were measured. Next, a part of fresh E. grandis leaves was weighed immediately to obtain the fresh weight (FW), the leaves were then soaked in deionized water for 24 h, the saturated weight (SW) was determined, and the leaves were dried for 48 h at 75°C to determine the dry weight (DW). The RWC in leaves was calculated as RWC = (FW 2 DW)/(SW 2 DW) Â 100%.
Analysis of active substances. The proline content was measured at 520 nm with a UV-visible (UVvis) spectrophotometer (Mapada, Shanghai, China) according to methods described previously by Bates et al. (80). Leaves and roots tissue (1 g) from fresh plant samples were weighed, frozen, and ground into a powder. One milliliter of an extract solution was added for ice bath homogenization, and the mixture was then centrifuged at 10,000 Â g at 4°C for 20 min. The contents of the active substances MDA, H 2 O 2 , and O 2 Á2 were then determined using an MDA kit, an H 2 O 2 kit, and an O 2 Á2 kit (Solarbio, Beijing, China), respectively, according to the manufacturer's protocol.
Analysis of antioxidant enzyme activity. Fresh E. grandis leaves or root tissues (about 1 g) were ground with 1 mL chilled buffer containing 50 mM potassium phosphate buffer (pH 7.8), 1 mM EDTA, 0.3% Triton X-100, and 1% polyvinylpyrrolidone. The mixture was then centrifuged at 10,000 Â g for 20 min at 4°C. Next, we conducted an analysis of the enzyme activity in the supernatant. Peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities were measured according to methods described previously by Beyer and Fridovich (81) and Amako et al. (82).
Phylogenetic analysis. According to the released genome of E. grandis (48), the MAPK cascade proteins of E. grandis were searched in the NCBI plant database using the BLAST program (83). The unrooted phylogenetic tree of E. grandis MAPK cascade proteins was generated using the neighbor-joining method in the MEGA11 program. The multiple-sequence alignment was performed using ClustalW, and the evolutionary distances were computed using the Poisson correction method. Bootstrap analysis was performed with 1,000 replicates. The motif distribution of MAPK cascade proteins in E. grandis was analyzed using an online website designed for this purpose (http://meme-suite.org/tools/meme), and a figure was created using tbtools (84).
RNA isolation, cDNA synthesis, RT-PCR, and qRT-PCR. Total RNA was extracted from E. grandis using the CTAB (cetyltrimethylammonium bromide) method (85), and first-strand cDNA synthesis was initiated using HiScript III reverse transcriptase (catalog number R323-01; Vazyme, Nanjing, China). Quantitative real-time PCRs were performed using ChamQ universal SYBR quantitative PCR (qPCR) master mix (Vazyme, Nanjing, China) in a 96-well real-time PCR system (Bio-Rad). All of the reactions were performed with three technical replicates of three biological replicates. The EgUBI3 genes from E. grandis and the RiEF1a gene from R. irregularis were used as the internal controls for normalization. The relative expression levels of the genes were computed by the 2 2DDCT method of relative quantification. A list of gene-specific primers used for qRT-PCR is given in Table S1 in the supplemental material.
Statistical analyses. All of the data were assayed using the SPSS Statistics 22 program. One-way analysis of variance (ANOVA) and Tukey's test were used to compare the differences among treatments. The data are presented as the means 6 standard errors (SE) for different replicates. Different letters in the figures indicate a significant difference at a P value of ,0.05. Redundancy analysis (RDA) was performed by using RStudio.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.