Constitutive Expression of Arabidopsis Senescence Associated Gene 101 in Brachypodium distachyon Enhances Resistance to Puccinia brachypodii and Magnaporthe oryzae

Brachypodium distachyon, as an effective model of cereal grains, is susceptible to most destructive cereal pathogens. Senescence associated gene 101 (SAG101) has been studied extensively in Arabidopsis. SAG101 is one of the important regulators of plant immunity. However, no homologous genes of AtSAG101 were found in B. distachyon. In this study, the AtSAG101 gene was transformed into B. distachyon. Three transgenic plant lines containing the AtSAG101 gene were confirmed by PCR and GUS gene activity. There were fewer Puccinia brachypodii urediospores in the AtSAG101-overexpressing plants compared to wild type plants. P. brachypodii biomass was obviously decreased in AtSAG101 transgenic plants. The length of infection hyphae and infection unit areas of P. brachypodii were significantly limited in transgenic plants. Moreover, there were small lesions in AtSAG101 transgenic plants challenged by Magnaporthe oryzae. Salicylic acid accumulation was significantly increased, which led to elevated pathogenesis-related gene expression in transgenic B. distachyon inoculated by P. brachypodii or M. oryzae compared to wild type plants. These results were consistent with infected phenotypes. Overexpression of AtSAG101 in B. distachyon caused resistance to M. oryzae and P. brachypodii. These results suggest that AtSAG101 could regulate plant resistance in B. distachyon.


Introduction
Plants are continually threatened by a wide variety of potential pathogens in the environment. To cope with pathogens, plants have evolved multiple defense mechanisms to avoid or limit infection [1]. Plants produce an inducible hormone to prevent pathogen invasion and reproduction. Salicylic acid (SA) is a common signal that is essential for resisting pathogens [2]. Salicylic acid (SA) accumulation induces the basal defense or plant resistance (R) gene-pathogen avirulence gene interactions, which induce immunity when host tissues respond to fungi pathogen invasion. An important role of SA is to induce pathogenesis-related (PR) genes expression [3].
Senescence associated gene 101 (SAG101), a 3 lipase-like defense regulator, is an essential component of plant basal resistance against pathogenic strains [4]. SAG101 regulates the production of

AtSAG101 Transgenic Plants Induce Resistance to P. brachypodii
To test the function of AtSAG101 during B. distachyon-P. brachypodii interactions, AtSAG101 transgenic plants were inoculated by the F-CO isolate of P. brachypodii. The B. distachyon Bd21-3 genotype is susceptible to P. brachypodii. Wild type (WT) plants produced numerous urediniospores 15 dpi after inoculation with F-CO. Compared to WT leaves, limited urediospore production was observed on leaves of AtSAG101 transgenic plants ( Figure 2A). P. brachypodii biomass was used to further verify phenotypes. P. brachypodii biomass was obviously decreased in the leaves of AtSAG101 transgenic plants compared to WT plants ( Figure 2B). brachypodii. Significant differences were determined using Student's t-test: *, p < 0.05. WT, wild type plants; L2-1, L3-2 and 5-2, transgenic B. distachyon plants.
Furthermore, histological changes in AtSAG101 transgenic plants inoculated with F-CO were observed ( Figure 3A). Length of infection hyphae (IH) and infection unit areas were observed and calculated. At 48 h after infection with the F-CO isolate, IH growth was significantly limited in AtSAG101 transgenic plants ( Figure 3B). Moreover, the P. brachypodii infection unit area was

AtSAG101 Transgenic Plants Induce Resistance to P. brachypodii
To test the function of AtSAG101 during B. distachyon-P. brachypodii interactions, AtSAG101 transgenic plants were inoculated by the F-CO isolate of P. brachypodii. The B. distachyon Bd21-3 genotype is susceptible to P. brachypodii. Wild type (WT) plants produced numerous urediniospores 15 dpi after inoculation with F-CO. Compared to WT leaves, limited urediospore production was observed on leaves of AtSAG101 transgenic plants ( Figure 2A). P. brachypodii biomass was used to further verify phenotypes. P. brachypodii biomass was obviously decreased in the leaves of AtSAG101 transgenic plants compared to WT plants ( Figure 2B).

AtSAG101 Transgenic Plants Induce Resistance to P. brachypodii
To test the function of AtSAG101 during B. distachyon-P. brachypodii interactions, AtSAG101 transgenic plants were inoculated by the F-CO isolate of P. brachypodii. The B. distachyon Bd21-3 genotype is susceptible to P. brachypodii. Wild type (WT) plants produced numerous urediniospores 15 dpi after inoculation with F-CO. Compared to WT leaves, limited urediospore production was observed on leaves of AtSAG101 transgenic plants ( Figure 2A). P. brachypodii biomass was used to further verify phenotypes. P. brachypodii biomass was obviously decreased in the leaves of AtSAG101 transgenic plants compared to WT plants ( Figure 2B). Furthermore, histological changes in AtSAG101 transgenic plants inoculated with F-CO were observed ( Figure 3A). Length of infection hyphae (IH) and infection unit areas were observed and calculated. At 48 h after infection with the F-CO isolate, IH growth was significantly limited in AtSAG101 transgenic plants ( Figure 3B). Moreover, the P. brachypodii infection unit area was Furthermore, histological changes in AtSAG101 transgenic plants inoculated with F-CO were observed ( Figure 3A). Length of infection hyphae (IH) and infection unit areas were observed and calculated. At 48 h after infection with the F-CO isolate, IH growth was significantly limited in AtSAG101 transgenic plants ( Figure 3B). Moreover, the P. brachypodii infection unit area was significantly reduced (P < 0.05) in AtSAG101 transgenic B. distachyon leaves at 120 hpi compared to WT leaves ( Figure 3C). Plants 2020, 9, x FOR PEER REVIEW 4 of 11 significantly reduced (P < 0.05) in AtSAG101 transgenic B. distachyon leaves at 120 hpi compared to WT leaves ( Figure 3C).

SA Levels Are Increased in AtSAG101-Overexpressing B. distachyon Leaves
SAG101 induces the production of SA to limit pathogen growth. To test a possible role of AtSAG101 in regulating SA, we examined SA levels in AtSAG101 transgenic B. distachyon leaves ( Figure 4A). SA accumulation was slightly higher in transgenic B. distachyon leaves compared to WT plants when unchallenged by P. brachypodii. At 24 hpi, SA accumulation was obviously increased (the range from 20.69 to 26.39 ng/g) in transgenic plants infected with P. brachypodii. However, there was no obvious change in SA accumulation in WT plants challenged by P. brachypodii, indicating a different response to that shown earlier in WT plants challenged by P. brachypodii.

SA Levels Are Increased in AtSAG101-Overexpressing B. distachyon Leaves
SAG101 induces the production of SA to limit pathogen growth. To test a possible role of AtSAG101 in regulating SA, we examined SA levels in AtSAG101 transgenic B. distachyon leaves ( Figure 4A). SA accumulation was slightly higher in transgenic B. distachyon leaves compared to WT plants when unchallenged by P. brachypodii. At 24 hpi, SA accumulation was obviously increased (the range from 20.69 to 26.39 ng/g) in transgenic plants infected with P. brachypodii. However, there was no obvious change in SA accumulation in WT plants challenged by P. brachypodii, indicating a different response to that shown earlier in WT plants challenged by P. brachypodii.
To determine if the expression levels of defense-related genes were affected in the AtSAG101 transgenic plants after challenge by P. brachypodii, we selected PR genes for quantitative RT-PCR (qRT-PCR) analysis. The expression levels of PR1, and PR5 were significantly increased in transgenic B. distachyon leaves infected with P. brachypodii at 24, 48, and 120 hpi ( Figure 4B).
To determine if the expression levels of defense-related genes were affected in the AtSAG101 transgenic plants after challenge by P. brachypodii, we selected PR genes for quantitative RT-PCR (qRT-PCR) analysis. The expression levels of PR1, and PR5 were significantly increased in transgenic B. distachyon leaves infected with P. brachypodii at 24, 48, and 120 hpi ( Figure 4B).

AtSAG101 Transgenic Plants Produce Resistance to Magnaporthe oryzae
Magnaporthe oryzae is an important rice fungus that can infect B. distachyon. In this study, we also inoculated AtSAG101 transgenic plants with M. oryzae to test the function of AtSAG101 during B. distachyon-M. oryzae interactions ( Figure 5A). AtSAG101 transgenic plants showed resistance to disease symptoms with small lesions, while large disease lesions were observed in WT plants at 5 dpi. Moreover, there was a 2.2-fold peak in SA accumulation in transgenic B. distachyon leaves challenged by M. oryzae at 24 hpi ( Figure 5B). However, there was no obvious change in WT plants challenged with M. oryzae. The expression levels of PR1 and PR5 were significantly induced in transgenic plants challenged by M. oryzae at 24, 48, and 96 hpi ( Figure 5C).

Discussion
Plants utilize multilayered defense strategies to limit or resist pathogen infection [1]. The lipase-like proteins, enhanced disease susceptibility1 (EDS1), SAG101 and phytoalexin deficient 4 (PAD4), are important regulators of plant immunity [15,16]. EDS1 and PAD4 are present in monocots and eudicots according to phylogenetic analysis, whereas SAG101 is not found in monocot genomes [17,18]. We also did not find homologous genes of AtSAG101 in the B. distachyon genome, thereby inferring evolution of SAG101 in different plants. In this study, we transformed the AtSAG101 gene into B. distachyon to determine if overexpression of the AtSAG101 gene produces resistance to different pathogens. Three transgenic plant lines (Lines 2-1, 3-2 and 5-2) were successfully transformed plants containing the AtSAG101 gene, which was confirmed by PCR and the GUS phenotype.
SAG101 is an essential component of plant basal resistance. In Arabidopsis, SAG101 plays important roles in resistance to bacterial pathogens and turnip crinkle virus [6]. In N. benthamiana, NbSAG101a is involved in resistance to Xanthomonas campestris pv. Vesicatoria [7]. AtSAG101 induces resistance activity in N. benthamiana, inferring it may enhance resistance in other plants [19]. In this study, we demonstrated that overexpression of the AtSAG101 gene produces resistance to P. brachypodii and M. oryzae. After inoculation with the P. brachypodii F-CO isolate, limited urediospore production was observed on AtSAG101 transgenic plant leaves. The biomass and detailed histological analysis confirmed that IH and infection unit areas of P. brachypodii were inhibited in AtSAG101 transgenic plants. These results were consistent with infected phenotypes. Moreover, AtSAG101 transgenic plants showed resistance to disease symptoms with small disease lesions compared to WT plants during B. distachyon-M. oryzae interactions. These results indicated that AtSAG101 may regulate immune signaling in different plants to multiply pathogenic strains.
SA, as an important plant defense signalling component, takes part in resistance to biotrophic pathogens. PAD4 is a key gene involved in pathogen-induced SA accumulation [20]. SAG101, which is thought to serve as a substitute for PAD4, functions in plant resistance [21]. There are different roles of PAD4 and SAG101 in regulating SA. Previously, we identified that SA accumulation is increased in AtPAD4 transgenic B. distachyon resistance to P. brachypodii [22]. However, SA accumulation is not obviously changed in WT plants and AtPAD4 transgenic plants lacking P. brachypodii infection. In this study, the SA levels were higher in AtSAG101 transgenic plants compared to WT plants. Moreover, SA accumulation was obviously increased when transgenic plants were challenged with P. brachypodii and M. oryzae. These results suggested that AtSAG101 transgenic plants enhance resistance to P. brachypodii and M. oryzae by increasing SA accumulation. We also found that PAD4 and SAG101 play different roles in mediating SA accumulation in B. distachyon. The resistance was not effectively activated in B. distachyon WT plants challenged with P. brachypodii and M. oryzae. SAG101 is not required for the SA-mediated induction of R genes [6]. We inferred overexpression of AtSAG101 can directly activate SA pathway in B. distachyon to involve in plant immunity, which is independent on the SA-mediated induction of R genes. However, the complex mechanism of AtSAG101 increasing SA accumulation needs to be studied further.
SA accumulation induces the expression of defense genes in response to virulent pathogens [23]. For example, the TaMAPK4 (a wheat MAP kinase 4) gene is involved in regulating SA accumulation. In TaMAPK4 knockdown plants, the expression levels of TaPR1 and TaPR5 are obviously reduced [24]. In this study, we found that the expression levels of PR1 and PR5 were significantly induced in AtSAG101 transgenic plants challenged by P. brachypodii and M. oryzae. Therefore, these results suggested that the accumulation of PR proteins may lead to enhanced disease resistance in AtSAG101-overexpressing plants.
In summary, three transgenic plant lines were successfully transformed to contain the AtSAG101 gene. We showed that overexpression of AtSAG101 in B. distachyon enhances resistance to M. oryzae and P. brachypodii. SA accumulation was increased in AtSAG101 transgenic plants challenged with B. distachyon, which lead to increased PR gene expression. Overexpression of AtSAG101 can directly activate plant defense. The results suggested that broad-spectrum genes can be selected in the process Plants 2020, 9, 1316 8 of 11 of breeding. In particular, those downstream resistance factors which are directly involved in regulating the resistance pathway are valuable candidates in disease resistance breeding.

Plant and Fungal Materials
In this study, the B. distachyon Bd21-3 genotype, P. brachypodii F-CO isolate and M. oryzae RO1-1 isolate were used. Bd21-3 is susceptible to RO1-1 and F-CO. B. distachyon was grown in a 22 • C growth chamber with 16 h (h) of light and 8 h of dark. Three seedlings were grown 8 cm diameter pots with medium, and 5 weeks old plants were inoculated with F-CO. Each treatment contains WT and three different transgenic lines, three plants for each line. Parallel mock inoculations were performed using tap water. Inoculation and incubation of F-CO followed the procedures as previously described [22]. Inoculated leaves were harvested at 0, 24, 48 and 96 h post inoculation (hpi). The inoculation results were evaluated at 15 dpi.
Five-week-old seedlings were challenged with M. oryzae spore (1 × 10 5 spores mL −1 ) by spraying. Seedlings inoculated by RO1-1 were kept in a humid chamber at 28 • C. Disease symptoms were evaluated at 5 dpi. Three independent biological replications were performed for each treatment.

Gene Cloning and Agrobacterium Transformation
Total RNA of A. thaliana was extracted using the TrizolTM Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. To synthesize cDNA from RNA, a Revert Aid First-strand cDNA synthesis kit from Fermentas (www.thermosscientific.com/fermentas) was used. The AtSAG101 gene (GenBank accession At5g14930, coding sequences without stop codon) was cloned from Arabidopsis leaves. A1611 bp PCR product was inserted into the pU1301 vector, which contains ubiquitin promotor and the reporter gene β-glucuronidase (GUS). The primers used for plasmid construction in the present study are listed in Supplementary Table S1. The pU1301-AtSAG101 vector was constructed for the transgenic plants using the Agrobacterium AGL1. All constructs were verified by sequencing and double digestion, and they were then transformed into B. distachyon following the procedure of Vogel [25]. Three independent homozygous T3 lines with a high expression level of AtSAG101 were selected for further study.

Histochemical Staining
Histochemical staining of leaves to detect GUS gene expression was conducted as previously described [24], except no chloramphenicol was used in the stain. Leaves were transferred into microtiter wells containing 500 µL of GUS staining solution (10 mM EDTA, 100 mM Na phosphate at pH 7, 1 mg/mL of X-Gluc, and 0.1% Triton X-100). Stained leaves were cleared in 95% ethanol to visualize localized staining. Sectioned samples were prepared for photography.

Endogenous SA Level Analysis
B. distachyon mutant and WT plant SA levels were analyzed by HPLC-MS [25]. The SA extraction was performed as previously described [22]. 250 mg of the frozen tissue was extracted and quantitated for SA. SA was extracted with MeOH-H 2 O-HOAc (90:9:1, v/v), the extract was evaporated and injected into liquid chromatography-electrospray ionization tandem mass spectrometry system (API 2000; AB SCIEX, United States of America (USA)). Standards of SA >99% (Fluka, Buchs, Switzerland) was made by diluting the standard solutions with the initial LC mobile phase (0.05% HOAc in H 2 O-MeCN, 85:15, v/v). SA quantitation was analyzed using the standard addition method of SA solutions (range of 50 to 1000 ng/mL). SA concentrations were calculated according to the detection results.

Histological Observation of Fungal Growth
P. brachypodii growth and development in mutant or wild type plants were characterized by histopathological analysis. Inoculated leaves were sampled at 48 h and 120 h, and leaves were treated and stained as previously described [25]. In brief, leaves were cut to 1.5 cm segments, which were fixed and decolorized in ethanol/trichloromethane (3:1 v/v) containing 0.15% (w/v) trichloroacetic acid for 3-5 days. For microscopic observation, leaves were treated and stained with WGA (wheat germ agglutinin conjugated to Alexa-488) (Invitrogen., Carlsbad, CA, USA). The lesion area and length were observed by an Olympus BX-53 microscope (Olympus Corp., Tokyo, Japan) and measured using CellScan Entry software. At least 30 randomly selected infection sites were measured, and 5 segments were randomly selected per treatment. Statistical analysis was performed using IBM SPSS 19 software (SPSS Inc., Chicago, IL, USA) with standard deviations and Tukey's test.

PCR Analysis
qRT-PCR reactions of 25 µL included 25 ng cDNA, 10 µL Taq Mix and 0.5 µM of each primer. PCR conditions were as follows: 95 • C for 1 min, 30 times (95 • C for 20 s, 58 • C for 15 s, and 72 • C for 1 min), and 72 • C for 4min. To standardize the data, ubiquitin-conjugating enzyme 18 gene (UBC18) was the internal reference. qRT-PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems). Electrophoresis was performed using a 1.5% agarose gel. qRT-PCR data were analyzed by the comparative 2 −∆∆Ct method [26].

Biomass Analysis
To estimate changes in fungal biomass, the DNA level of P. brachypodii EF1 was measured by quantitative PCR with the DNA level of reference gene B. distachyon EF1. Relative fungal growth was then calculated as a ratio (Pb-EF1/Bd-EF1) to reflect the amplification efficiency.  Table  S1: Primers for plasmid constructions and PCR.