- © 2019 American Society of Plant Biologists. All rights reserved.
Abstract
To defend against pathogens, plants have developed complex immune systems, including plasma membrane receptors that recognize pathogen-associated molecular patterns, such as chitin from fungal cell walls, and mount a defense response. Here, we identify a chitinase, MoChia1 (Magnaporthe oryzae chitinase 1), secreted by M. oryzae, a fungal pathogen of rice (Oryza sativa). MoChia1 can trigger plant defense responses, and expression of MoChia1 under an inducible promoter in rice enhances its resistance to M. oryzae. MoChia1 is a functional chitinase required for M. oryzae growth and development; knocking out MoChia1 significantly reduced the virulence of the fungus, and we found that MoChia1 binds chitin to suppress the chitin-triggered plant immune response. However, the rice tetratricopeptide repeat protein OsTPR1 interacts with MoChia1 in the rice apoplast. OsTPR1 competitively binds MoChia1, thereby allowing the accumulation of free chitin and re-establishing the immune response. Overexpressing OsTPR1 in rice plants resulted in elevated levels of reactive oxygen species during M. oryzae infection. Our data demonstrate that rice plants not only recognize MoChia1, but also use OsTPR to counteract the function of this fungal chitinase and regain immunity.
INTRODUCTION
Plants are under constant attack from various microbial pathogens. Many phytopathogens colonize the plant apoplast, particularly the biotrophic pathogens and hemibiotrophic pathogens during their biotrophic stage (Jashni et al., 2015; Cao et al., 2016). Plants have developed a sophisticated immune system that recognizes the presence of pathogens by their conserved features. The plasma membrane-located pathogen recognition receptors (PRRs) contain extracellular domains that can bind and recognize various pathogen-associated molecular patterns (PAMPs). Upon activation, the PRRs transduce signals to downstream components to initiate the immune responses (Zipfel, 2014; Boutrot and Zipfel, 2017), which typically involves mitogen-activated protein kinase (MAPK) signaling, callose deposition, a reactive oxygen species (ROS) burst, and pathogen-related gene expression, referred to as PAMP-triggered immunity (PTI; Jones and Dangl, 2006; Zipfel, 2014).
Two common PAMPs are bacterial flagellin and the elongation factor-Tu (EF-Tu), perceived in Arabidopsis (Arabidopsis thaliana) by FLAGELLIN-SENSITIVE2 (FLS2) and EF-Tu receptor, respectively (Jones and Dangl, 2006; Zipfel, 2014). The recognition of these PAMPs can trigger PTI. During infection by a filamentous fungus, chitin, the key constituent of the fungal cell wall, is recognized by plant receptors categorized as lysin motif receptor-like kinases. Chitin is the most abundant amino polysaccharide in the biosphere and is composed of polymeric N-acetylglucosamine (GlcNAc; Langner and Göhre, 2016). In Arabidopsis, the lysin motif receptor-like kinases chitin-elicitor receptor kinase 1 (AtCERK1) and lysin motif receptor kinase 5 form a receptor complex that recognizes chitin (Cao et al., 2014); however, in rice (Oryza sativa), the LysM domain of chitin-elicitor binding protein (CEBiP) binds chitin and associates with OsCERK1, which phosphorylates downstream receptor-like cytoplasmic kinase 185 (OsRLCK185) to activate intracellular immune pathways (Yamaguchi et al., 2013).
Successful pathogens must overcome host immunity to colonize the apoplast, either by avoiding the activation of, or directly suppressing, PTI. Pep1 (Protein essential during penetration 1), an effector of the maize smut fungus (Ustilago maydis), directly interferes with the generation of ROS in the host apoplast by inhibiting peroxidase activity (Hemetsberger et al., 2012). In Cladosporium fulvum, the effector protein Ecp6 (extracellular protein 6) possesses LysM domains and sequesters fungal chitin oligomers to avoid chitin triggering immunity in the host plant (de Jonge et al., 2010). Similarly, Magnaporthe oryzae secretes Secreted LysM Protein 1 (Slp1), a LysM-containing effector protein, to compete with OsCEBiP for chitin binding, thereby preventing the activation of PTI in rice (Mentlak et al., 2012). In addition to circumventing the extracellular perception by the PRRs, many pathogens deliver effectors into the plant cells, which perturb multiple signaling pathways in the host, including subverting host apoplastic immunity by reducing the ROS burst, callose deposition, and other PTI responses. The bacterial pathogen Pseudomonas syringae secretes an effector protein AvrPto to block FLS2 phosphorylation (Xiang et al., 2008), and the other effector, AvrPtoB, targets FLS2 and CERK1 for degradation (Göhre et al., 2008; Gimenez-Ibanez et al., 2009).
In addition to the well-characterized PAMPs flagellin, EF-Tu, and chitin, many pathogen-secreted metabolites or virulence proteins can also activate the plant immune system; these include lipopolysaccharides, peptidoglycan volatiles, glycoproteins, cell wall degradation enzymes (CWDE), and other pathogen-secreted proteins (Liu et al., 2012; Ranf et al., 2015). Proteins that are secreted into the plant apoplast by pathogens are believed to play a fundamental role in plant apoplastic immunity (Jashni et al., 2015); for example, the pathogen-secreted CWDEs primarily target and modulate plant cell walls to damage the cell’s integrity. Plants, on the other hand, secrete diverse proteases or protease inhibitors into the apoplast, presumably suppressing the activities of pathogen-derived CWDEs or other virulence proteins (Rooney et al., 2005; Jashni et al., 2015).
Host–pathogen interactions in the plant apoplast may be elegantly regulated. One recent example is the interaction of the oomycete pathogen Phytophthora sojae and its host plant soybean (Glycine max; Ma et al., 2017). The pathogen secretes an apoplastic xyloglucan-specific endoglucanase (PsXEG1) that facilitates infection, but its activity could potentially be inhibited by the soybean-derived apoplastic glucanase inhibitor protein, GmGIP1; however, P. sojae secretes a paralogous nonfunctional PsXEG1-like protein, PsXLP1, that acts as a decoy, binding to GmGIP1 with higher affinity than PsXEG1, thereby freeing the endoglucanase to support the infection (Ma et al., 2017). This finding suggests the importance of apoplast decoys in the soybean response to pathogen invasion; however, this strategy has not yet been observed in other plant–pathogen interactions.
Fungal pathogens harbor multiple chitinases that continuously remodel their polymeric chitin and ensure the plasticity of their cell walls during growth and infection (Langner and Göhre, 2016). Chitinases are essential for cell separation in U. maydis (Langner et al., 2015). However, the endochitinase of Verticillium dahlia inhibits early fungal spore germination and triggers immune responses in Arabidopsis and cotton (Gossypium hirsutum; Cheng et al., 2017). In this study, we report that MoChia1 (M. oryzae chitinase 1), a chitinase from the rice blast pathogen M. oryzae, can activate the immune response in the rice apoplast. MoChia1 binds chitin and suppresses the chitin-triggered ROS burst in rice; however, the rice plants secrete a tetratricopeptide-repeat protein, OsTPR1, which prevents MoChia1 from binding chitin in the apoplast, thereby rescuing the chitin-triggered ROS burst and regaining immunity.
RESULTS
Identification of MoChia1
We previously predicted that M. oryzae secretes over 150 effector proteins into the rice apoplast, many of which target plant cell wall components and are therefore unlikely to be delivered into the intracellular space during infection (Cao et al., 2017). Kim et al. identified over 400 proteins secreted by M. oryzae into the rice apoplast during infection, which could potentially be perceived by the rice cells as “non-self” fungal proteins (Kim et al., 2013). To screen the fungus-secreted proteins for new elicitors/PAMPs that trigger the plant immune response, we used fast protein liquid chromatography (LC) to isolate and purify the proteins that induced a ROS burst in rice suspension cells. After molecular exclusion and purification using anion exchange, we isolated a fraction that could significantly activate the ROS burst (Supplemental Figures 1A to 1D). Further protein identification using LC-MS/MS led to the discovery of five proteins in total (Supplemental Table 1).
We next expressed these proteins and tested whether they could induce the ROS burst in rice cells. The sequences encoding these proteins were fused with the Escherichia coli gene encoding the maltose binding protein (MBP). Four recombinant proteins were produced in E. coli (the recombinant MGG-04436 could not be expressed). The proteins were purified and then tested for their ability to cause a ROS burst in a suspension of rice cells. Only one of the proteins, MGG_08054, activated a strong ROS burst (Supplemental Figures 1E and 1F). MGG_08054 is a putative chitinase, and was named MoChia1. Notably, the MoChia1-triggered ROS burst is much slower than that caused by previously identified PAMPs, such as chitin or flg22 (Figure 1A). MoChia1 is phylogenetically distant from its closest orthologs in M. oryzae (Supplemental Figure 2; Supplemental Data Set).
MoChia1 Activates Pathogen-Triggered Immune Responses in Rice.
(A) MoChia1 activates the ROS burst in rice suspension cells. Recombinant MBP-MoChia1 protein was purified from E. coli, and 2 μg/ml protein was used for the luminol-based ROS burst assay. Flg22 (100 nM) and chitin (0.1 μg/ml) served as positive controls. MBP served as a negative control. Values are means ± sd (n = 4). RLU, relative light units.
(B) MoChia1 protein can activate MAP kinase signaling in rice. MoChia1 protein (1 μg/ml) was incubated with rice suspension cells. MBP protein (1 μg/ml) was used as the negative control. Activated MAPKs were detected by immunoblotting with the phospho-p38 MAPK antibody at the indicated times. The corresponding bands are represented for the phosphorylation of mitogen-activated protein kinase 3 and mitogen-activated protein kinase 6. Coomassie brilliant blue (CBB) staining of ribulose-1,5-bis-phosphate carboxylase/oxygenase was used to ensure equal loading in each lane. The experiment was repeated three times with similar results.
(C) MoChia1 induces callose deposition in rice. The leaves of 2-week-old rice seedlings were treated with 0.6 μg/ml MoChia1 or 8 nM chitin for 16 h, and then stained using aniline blue. MBP served as the mock treatment.
(D) The overexpression of MoChia1 leads to an autoimmune response in rice. MoChia1, driven by the Ubiquitin promoter, was ligated into the pC1390U binary vector and transformed into the rice variety Nipponbare (wild type). Lines 5 and 8 are two representative independent lines. Bar = 1 cm.
(E) MoChia1 overexpression leads to ROS accumulation in rice. Two-week-old DEX:MoChia1 plants were treated with 30 μM DEX for 24 h; then the leaves were stained with DAB. Lines 4 and 6 are two independent transgenic lines. Bar = 1 cm.
(F) MoChia1 is a functional chitinase, and the Glu137 mutation abolishes its enzymatic activity. The chitinase enzyme activity of the recombinant MBP-MoChia1 protein was analyzed, using colloid chitin as substrate. The reaction of DNS (3,5-dinitrosalicylic acid) and GlcNAc was monitored at OD 565 nm. One unit of chitinase activity was defined as the amount of enzyme required to release 1 μmol of N-acetyl-d-glucosamine per hour. Values are means ± sd (n = 3). **Significant differences from MBP at P < 0.01 (Student’s t test).
(G) The enzyme activity of MoChia1 is not required for the ROS burst. Rice suspension cells were used to measure ROS production in a luminol-based assay, using 1 μg/ml protein. MBP was used as a negative control. Values are means ± sd (n = 4).
MoChia1 Functions as a PAMP
Because MoChia1 can activate a ROS burst in rice cells, we speculated that this protein acts as an elicitor/PAMP during rice blast infection. MoChia1 was found to be secreted during mycelial growth, and the secretion depends on secretion signal peptide (SP), because removal of SP (MoChia1NSP) abolishes MoChia1 secretion to the supernatant (Supplemental Figure 3A). Classical PAMPs induce MAPK signaling and callose deposition (Luo et al., 2017). When MoChia1 was exogenously applied to rice cells, we found that the MAPK pathway was significantly activated, whereas the mock treatment did not activate this pathway (Figure 1B). In addition, MoChia1 induced callose deposition in the rice cell walls, similar to the chitin treatment (Figure 1C). We therefore concluded that MoChia1 is an active PAMP for rice plants.
To further study its role in planta, we expressed MoChia1 in rice; however, the constitutive expression of MoChia1 driven by the Ubiquitin promoter resulted in a dwarf phenotype and the eventual death of the transgenic plants (Figure 1D), indicating that MoChia1 may cause an autoimmune response in transgenic plants. We therefore used a dexamethasone (DEX)-inducible promoter (Luo et al., 2017) and generated the DEX:MoChia1 plants. As expected, MoChia1 was mainly localized in the apoplasts of the transgenic rice plants; however, the rice transcription factor RERJ1, which has been reported to be localized in the nucleus (Miyamoto et al., 2013), cannot be detected in the rice apoplasts (Supplemental Figure 3B). To investigate whether MoChia1 triggers ROS production in vivo, we examined the ROS levels of the transgenic plants using 3,3-diaminobenzidine (DAB) staining, revealing that more H2O2 accumulated in the DEX:MoChia1 plants following treatment with 30 µM DEX (Figure 1E). This result is consistent with the in vitro assays using rice suspension cells (Figure 1A) and demonstrates that MoChia1 can activate ROS accumulation in planta. Notably, the major PRR genes, except for OsFLS2, were not significantly induced in Dex-treated DEX:MoChia1 plants (Supplemental Figure 4A), suggesting that the MoChia1-triggered immune response is likely independent of known PRRs.
MoChia1 Chitinase Activity is Dispensable for the ROS Burst in Rice
Because MoChia1 is phylogenetically distant from other putative chitinases in the M. oryzae genome (Supplemental Figure 2), we investigated whether this protein is a functional chitinase by examining its enzymatic activity using colloidal chitin as the substrate (Niu et al., 2016). The recombinant MoChia1 could effectively hydrolyze chitin into monomeric GlcNAc (Figure 1F), indicating that this protein is a functional chitinase. Sequence alignment of the chitinase family proteins suggested that Glu137 is a conserved amino acid residue among chitinases, which was previously reported to be essential for chitinase activity (Supplemental Figure 4B; Lienemann et al., 2009). We therefore mutated the Glu137 (E) of MoChia1 to Gln (Q; MoChia1E137Q); this mutation abolished its chitinase activity (Figure 1F). Nevertheless, we found that chitinase activity is not required for MoChia1 to induce the ROS burst in rice cells, because MoChia1E137Q could still activate the ROS burst (Figure 1G).
Furthermore, we generated the MoChia1E137Q transgenic rice plants with both Ubiquitin and Dex-inducible promoters. The constitutive expression of MoChia1E137Q led to an autoimmune response in rice, as the transgenic plants showed a dwarf phenotype and eventual death (Supplemental Figure 4C). The DAB staining assay also revealed that 30 µM DEX-treated DEX:MoChia1E137Q plants accumulated more H2O2 in comparison with the untreated plants (Supplemental Figure 4D). These results demonstrate that the chitinase activity is not required for MoChia1-activated ROS accumulation in planta. By contrast, rice chitinases could not activate the ROS burst in rice cells (Supplemental Figures 5A and 5B), implying that plants have evolved a mechanism to avoid activation of the immune response by their own chitinases.
MoChia1 Plays a Fundamental Role in Fungal Growth
Because MoChia1 is a functional chitinase, we checked whether it is indeed required for fungal growth. MoChia1 was found to be differentially expressed in various fungal tissues (Figure 2A); for example, the expression of MoChia1 was higher in the mycelia and appressoria. Interestingly, during infection, MoChia1 was not expressed at high levels until 48 h post inoculation (hpi; Figure 2B).
MoChia1 is Required for the Growth and Development of M. oryzae.
(A) MoChia1 expression levels in different tissue or developmental stages. MoChia1 expression was investigated using RT-qPCR. The fungi were grown in CM media. Values are means ± sd (n = 3).
(B) MoChia1 expression levels during M. oryzae infection in rice. The rice leaves were inoculated with M. oryzae spores at a concentration of 1 × 105 per mL. The leaves were sampled at the indicated time points for RT-qPCR assays. Values are means ± sd (n = 3).
(C) MoChia1 affects mycelial growth. The images were taken from the wild-type Guy11 and ΔMoChia1 mutants grown on the CM medium for 2 weeks. Bar = 1 cm.
(D) Statistical analysis of the growth rate of mycelia in (C). Values are means ± sd (n = 8). **Significant differences from wild type at P < 0.01 (Student’s t test).
(E) ΔMoChia1 mutants produce fewer conidia. Conidia were observed under a light microscope after illumination for 24 h. The experiments were repeated three times with similar results.
(F) ΔMoChia1 mutants are less sensitive to cell wall stress. The Guy11, ΔMoChia1 mutants, and complementation strains were grown on complete medium containing 0.1% Congo Red. Photographs were taken after 7 d in culture at 28°C. Bar = 1 cm.
(G) ΔMoChia1 mutants display chitin accumulation in the mycelia and conidia. The chitin contents of the Guy11 and ΔMoChia1 mycelia were measured after a 2-d culture at 28°C. Fresh conidia after light induction were used for chitin content measurements. DW, dry weight. Values are means ± sd (n = 3). **Significant differences from Guy11 at P < 0.01 (Student’s t test).
(H) and (I) Knocking out MoChia1 leads to abnormal chitin deposition in the mycelia, conidia, and germination tubes. Mycelia were cultured for 14 days at 28°C. Mycelia, fresh conidia, and germinated conidia were used for cell wall staining with calcofluor. The fluorescence was observed under a fluorescence microscope using the DAPI channel. Bar = 10 μm.
Next, we knocked out MoChia1 in M. oryzae using the recombinant exchange method (Supplemental Figures 6A and 6B; Li et al., 2017). When grown on complete minimal (CM) media, the △MoChia1-2 mutant displayed a higher growth rate than the wild type (Figures 2C and 2D), whereas the growth rate of the fungal strain overexpressing MoChia1 was significantly reduced (Supplemental Figures 7A and 7B). The △MoChia1-2 mutants produced many fewer conidia than the wild type; however, this phenotypic defect could be rescued by complementation with wild-type MoChia1 (Figure 2E), indicating that MoChia1 plays an essential role in conidia production.
Because chitin is a major constituent of the fungal cell wall, we questioned whether M. oryzae could respond to cell wall stress treatments in the absence of MoChia1. We found that the △MoChia1-2 strain was resistant to treatment with 0.1% Congo Red, a cell wall stressor (Figure 2F), indicated by the larger diameter of its colony in comparison with the wild type after culturing for 7 d. These data suggest that MoChia1 may modify the fungal cell wall to regulate the response to cell wall stress. We then measured the chitin contents in the mycelia and conidia of the wild type and the △MoChia1 mutants; the two △MoChia1 mutant strains exhibited much higher chitin contents than the wild type in both the mycelia and conidia, indicating that MoChia1 has chitinase activity in vivo (Figure 2G). MoChia1 is also required for proper chitin deposition in the cell walls. As shown in Figures 2H and 2I, abnormal chitin deposition was observed. The fluorescence signal was found primarily in the tip of the mycelium of wild type, but was dispersed in the △MoChia1 mutants (Figure 2H), revealing that MoChia1 affects chitin distribution. In addition, the fluorescence signal was also found at the germination point of the germination tube (Figure 2H); however, in the △MoChia1 conidia cells it was found at the septal area. These data demonstrate that MoChia1 is a critical gene for modulating fungal growth and the response to cell wall stresses.
MoChia1 Contributes to M. oryzae Virulence
In general, developmental defects in fungi affect their virulence. We therefore evaluated the pathogenicity of △MoChia1 mutants as well as the MoChia1-OE strains on rice. On a hydrophobic glass surface, the △MoChia1 mutants exhibited much slower germination tube development and appressoria formation than the wild type (Figure 3A). At 3 hpi, the germination tube was almost completely developed in the wild type, and at 6 hpi, appressoria had formed; however, the development of these features was delayed by about 3 h in the △MoChia1 mutants. Accordingly, only 58.3% of the △MoChia1 mutants had formed appressoria at 6 hpi, but 91.2% of the wild-type fungi had done so (Figures 3B and 3C). Nevertheless, we found that the fungal hyphae of the △MoChia1 mutants had developed normally when observed at 24 and 48 hpi; however, the mutant hyphae grew more slowly, with a delay of ∼12 h compared with the wild type (Figure 3D). This slow development also largely reduced the pathogenicity of the △MoChia1 mutants on rice leaves following spray inoculation (Figures 3E and 3F), indicated by fewer lesions than were observed following inoculation with the wild-type strain. By contrast, the MoChia1 overexpression strains showed lower chitin levels than the control and the MoChia1E137Q overexpression strains (Supplemental Figure 7C). Interestingly, the MoChia1 overexpression strains also displayed lower virulence than the wild-type Guy11 (Supplemental Figures 7D and 7E).
MoChia1 is Required for M. oryzae Virulence.
(A) Germination of conidial spores of the wild type and ΔMoChia1 mutants. Conidia were germinated on glass cover slips. The germination tubes and appressorium development were observed at the indicated time points. ΔMoChia1-2 and ΔMoChia1-11 are two independent mutant strains. Bar = 10 μm.
(B) Appressorium formation is largely delayed in the ΔMoChia1 mutants. Appressorium development was observed after a 6-h incubation on a hydrophobic glass surface. Bar = 100 μm.
(C) Statistical analysis of appressorium formation in the wild type and ΔMoChia1 mutants observed in (B). Values are means ± sd (n = 3). **Significant differences from the wild type at P < 0.01 (Student’s t test).
(D) ΔMoChia1 display reduced virulence on rice. The rice leaf sheath was inoculated with a conidial suspension at a concentration of 1 × 105 conidia per mL in 0.2% Tween 20. The hyphae images were taken at 24 and 48 hpi. Bar = 10 μm.
(E) Disease symptoms of rice leaves infected with the wild-type M. oryzae, ΔMoChia1 mutants, and the complementation strains. Conidial suspensions (1 × 105 conidia per mL in 0.2% Tween 20) were sprayed onto the leaf surfaces of 2-week-old seedlings. Images were taken at 7 dpi (days post inoculation). Bar = 1 cm.
(F) Relative fungal biomass for (E). The fungal biomass was determined using qPCR of the M. oryzae Pot2 gene against the rice OsUbi1 gene. Values are means ± sd (n = 4). **Significant differences from the wild type at P < 0.01 (Student’s t test).
Melanin is known to contribute to fungal pathogenicity (Soanes et al., 2012). Knocking out MoChia1 remarkably reduced melanin accumulation in the fungus (Supplemental Figure 7F), and decreased the expression levels of the melanin biosynthesis-related genes RSY1 and 4HNR; however, the expression of AIB1 and BUF1 was not affected (Supplemental Figure 7G; Kawamura et al., 1997). In addition, the infection of rice cells with the △MoChia1 mutant fungus activated stronger expression of RbohB and RbohD, the defense-related marker genes, than infection with the wild-type strain (Supplemental Figures 8A and 8B). The above data imply that the loss of MoChia1 function reduces pathogen virulence because of the slower development of the hyphae, which is at least partially mediated by the enhanced immune response in rice.
Ectopic Expression of MoChia1 Activates the Immune Response
The data described above show that transiently induced MoChia1 expression leads to ROS accumulation in rice plants (Figures 1D and 1E). In addition, MoChia1 acts as a typical PAMP for rice plants (Figures 1B and 1C). We therefore investigated whether the ectopic expression of MoChia1 in rice could enhance the disease resistance of these plants. Because the constitutive expression of MoChia1 in rice caused an autoimmune response, we tested the disease response of the DEX:MoChia1. The DEX:MoChia1 plants showed enhanced resistance to M. oryzae when pretreated with 30 μM DEX (Figures 4A and 4B).
Ectopic Expression of MoChia1 Enhances Rice Resistance to M. oryzae.
(A) DEX:MoChia1 plants exhibit enhanced disease resistance to M. oryzae after DEX treatment. Two-week-old DEX:MoChia1 plants were treated with 30 μM DEX applied to the roots. After 24 h, the seedlings were spray-inoculated with conidial suspensions (1 × 105 conidia per mL in 0.2% Tween 20). The images were taken at 5 dpi. Bar = 1 cm.
(B) Relative fungal biomass in (A). Values are means ± sd (n = 4). **Significant differences from wild type at P < 0.01 (Student’s t test).
(C) The DEX:MoChia1 plants displayed enhanced disease resistance to M. oryzae after DEX treatment. Rice leaf sheaths were inoculated with conidial suspension at a concentration of 1 × 105 conidia per mL in 0.2% Tween 20. The hyphae images were taken at 24 and 48 hpi, respectively. Bar = 10 μm.
(D) DEX:MoChia1 plants exhibit enhanced ROS accumulation during M. oryzae infection after DEX treatment. The rice leaf sheath was inoculated with a conidial suspension at a concentration of 1 × 105 conidia per mL in 0.2% Tween 20. The DAB staining was performed at 24 and 48 hpi, respectively. Bar = 10 μm.
(E) and (F) OsPR10 and OsRbohA expression was elevated in DEX:MoChia1 plants. Two-week-old wild type and DEX:MoChia1 plants were pretreated with 30 μM DEX. After 24 h, RT-qPCR was used to examine the gene transcription levels in plants. Values are means ± sd (n = 4). **Significant differences from wild type at P < 0.01 (Student’s t test).
We next examined the infection process at the cellular level using the rice leaf sheath. The inoculation of the leaf sheath revealed that the induced ectopic expression of MoChia1 in rice cells restricted the mycelial growth of the pathogen (Figure 4C). Using DAB staining, we found elevated H2O2 levels in the infected cells of the DEX:MoChia1 plants compared with the wild type, which may result in the enhanced disease resistance of these plants (Figure 4D). We also found that the expression of OsPR10 and OsRbohA was significantly upregulated in the DEX-treated DEX:MoChia1 plants (Figures 4E and 4F). These genes are indicators of the activation of the immune response in rice plants (Yang et al., 2017), suggesting that MoChia1 leads to the activation of the immune response. These data demonstrate that the ectopic expression of MoChia1 can enhance blast disease resistance in rice.
MoChia1 Interacts with OsTPR1 in the Rice Apoplast
We attempted to identify the proteins that mediate MoChia1 recognition in the rice apoplast, or proteases in rice that could potentially degrade MoChia1. To this end, we conducted a yeast two-hybrid screen against a rice cDNA library using MoChia1 as the bait. This approach did not lead to the identification of the MoChia1-interacting protease, nor PRRs; however, we did identify one of proteins that can interact with MoChia1, OsTPR1. This protein is a member of the tetratricopeptide-repeat (TPR) family, which often function as scaffold proteins in protein–protein interactions (Cerveny et al., 2013). We confirmed the interaction of MoChia1 and OsTPR1 by expressing their full-length cDNA sequences in yeast (Figure 5A). Notably, the MoChia1E137 mutation did not affect the interaction (Figure 5A). The OsTPR1 protein contains three TPR motifs from amino acids 64 to 172 (Supplemental Figure 9A). We further identified the interaction region of OsTPR1 and found that the C-terminal of OsTPR1 was likely responsible for the interaction (Supplemental Figures 9A and 9B). GST pull-down assays using the recombinant GST-MoChia1 and MBP-OsTPR1 showed that the two proteins interacted with each other in vitro (Figure 5B). Because the sequence of MoChia128-370 is predicted to be a carbohydrate binding domain (CBD), we then tested the interaction between MoChia1 CBD and OsTPR1 by pull-down assays. We confirmed that MoChia1 CBD interacted with OsTPR1 (Figure 5B). We also tested their interaction in planta. MoChia1 was found to interact with OsTPR1 in Nicotiana benthamiana leaves using both split-luciferase and bimolecular fluorescence complementation (BiFC) assays (Figures 5C to 5E). The above results confirm the interaction of MoChia1 and OsTPR1 in vitro and in vivo.
MoChia1 Interacts with OsTPR1 In Vitro and In Vivo.
(A) MoChia1 and MoChia1E137Q interact with OsTPR1 in yeast. AD-MoChia1, AD-MoChia1E137Q, and BD-OsTPR1 plasmids were cotransformed into yeast cells and screened on synthetic dextrose media lacking Leu and Trp (SD-2). The single colonies were serially diluted onto SD-2 and SD-3 (synthetic dextrose media lacking Leu, Trp, and His) to observe the yeast cell growth. Yeast cotransformed with pGADT7-T+pGBKT7-53 served as a positive control. Yeast cotransformed with pGADT7-T+pGBKT7-lam served as a negative control. EV, empty vector.
(B) MoChia1 and its CBD interact with OsTPR1, revealed using GST pull-down assays. The recombinant MBP-OsTPR1, GST-MoChia1, and GST-MoChia1CBD proteins purified from E. coli were subjected to a GST pull-down analysis. Interacting proteins were visualized with immunoblotting.
(C) MoChia1 interacts with OsTPR1 in N. benthamiana, revealed using split luciferase assays. N. benthamiana leaves were co-infiltrated with 35S:MoChia1-nLUC and 35S:cLUC-OsTPR1. Luciferase complementation imaging assays were performed 2 d later. The gels at the right show the expression of the respective proteins. This experiment was repeated three times with similar results.
(D) MoChia1 but not MoChia1NSP interacts with OsTPR1 in N. benthamiana, revealed using split YFP assays. The experimental procedure was similar to that used in (C). MoChia1 or MoChia1NSP was fused with cYFP at the C terminus, and OsTPR1 was fused with nYFP at the N terminus. The images were observed under a confocal microscope 2 d later. Bar = 50 μm.
(E) Immunoblotting showed the expression of respective proteins in (D).
(F) OsTPR1 is localized to the plasma membrane in rice protoplasts. OsTPR1-GFP and the plasma membrane marker PCD-1002-CFP were co-expressed in rice protoplasts and visualized by confocal microscopy. GFP co-expressed with PCD-1002-CFP was the negative control. PCD-1002-CFP was assigned the pseudocolor red. Bar = 10 μm.
(G) OsTPR1 is localized to the plasma membrane in N. benthamiana. Proteins fused with respective fluorescence proteins were transiently expressed in N. benthamiana leaves following Agrobacterium-mediated transformation. Plasmolysis was performed by treatments with 10 mM NaCl. The images were captured using a confocal microscope. PCD-1002-CFP was assigned the pseudocolor red. Bar = 25 μm.
(H) MoChia1 is located in the plant apoplast. Gossypium hirsutum apoplastic peroxidase GhPOD10 and MoChia1-GFP were expressed in N. benthamiana leaves. The protein subcellular localization was observed using a confocal microscope. Top panel, protein localization before plasmolysis; bottom panel, protein localization after plasmolysis. The fluorescence curves were obtained following the direction of the white arrows. Bar = 25 μm.
It is worth noting that MoChia1 apoplast localization is essential for the interaction of MoChia1 and OsTPR1, because removal of MoChia1 SP (MoChia1NSP) abolished the interaction (Figures 5D and 5E). In addition, it is necessary to check the subcellular location of OsTPR1. OsTPR1 was found to colocalize with the plasma membrane marker PCD-1002 in rice protoplasts as well as in N. benthamiana leaves (Figures 5F and 5G; Du et al., 2013). The plasmolysis assay further supports that OsTPR1 localizes to the rice plasma membrane (Figure 5G). To investigate whether there is any extracellular domain/region(s), we used protease protection assays to explore the region. Both trypsin and protease K could degrade OsTPR1 but not green fluorescent protein (GFP) or ACTIN in rice protoplasts and N. benthamiana leaves (Supplemental Figures 9C to 9E), indicating that OsTPR1 carries extracellular domain/region(s). With use of the plasmolysis assay, the MoChia1 subcellular localization was determined, which was found in the plant apoplast following its transient expression in N. benthamiana leaves, similar to the reported apoplastic anionic gaiacol peroxidase of G. hirsutum (GhPOD10; Figure 5H; Li et al., 2016). The results also showed that, before plasmolysis, the MoChia1 and GhPOD10 colocalized with PCD-1002; however, after plasmolysis, they were separated from PCD-1002 and were localized in the plant apoplast (Figure 5H). The evidence suggests that MoChia1 likely interacts with OsTPR1 in the plant apoplast.
MoChia1 Suppresses the Chitin-Triggered ROS Burst in Rice, Whereas OsTPR1 Removes this Suppression
As described above, MoChia1 likely interacts with OsTPR1 in the rice apoplast during M. oryzae infection. We therefore investigated the biological significance of this interaction. MoChia1 is a chitinase and uses chitin as its substrate; MoChia1 as well as MoChia1E137Q were able to bind chitin in vitro in a chitin pull-down assay (Figure 6A), indicating that the chitinase activity is dispensable for binding chitin. OsTPR1 can also bind MoChia1; therefore, we investigated whether it competes with chitin to bind MoChia1. The result shows that less MoChia1 is pulled down in the chitin pull-down assay following the addition of increasing amounts of OsTPR1 (Figure 6A), indicating that OsTPR1 can interfere with the interaction between MoChia1 and chitin.
OsTPR1 Removes the MoChia1-mediated Suppression of the Chitin-Triggered ROS Burst.
(A) OsTPR1 interferes with the ability of MoChia1 to bind chitin. Left: Chitinase activity is dispensable for binding chitin. Right: Colloid chitin and purified recombinant proteins were used for chitin pull-down assays. Each 50-μL reaction mixture contained 50 μg colloid chitin and 2 μg MBP-MoChia1. GST-OsTPR1 protein (0, 40, and 80 μg) was added to the mixture and incubated for 1 h with constant shaking. The chitin-associated MBP-MoChia1 was detected using immunoblotting. The experiment was repeated three times with similar results.
(B) MST assays show that MoChia1 interacts with OsTPR1. The recombinant proteins were contained in NT standard capillaries. The solid curve is the fit of the data points to the standard Kd-fit function. The experiment was repeated at least three times with similar results. Kd, dissociation constant. Bars ± sd (n = 3).
(C) OsTPR1 competes with chitin for MoChia1 binding, as revealed using MST assays. The experimental procedure was the same as in (B).
(D) MoChia1 suppresses the chitin-triggered ROS burst in rice cells, whereas OsTPR1 removes this suppression. A 1 μg/ml MoChia1 or 1 μg/ml OsTPR1 aliquot was incubated with 0.2 μg/ml chitin in the buffer [50 mM Tris-HCl (pH 7.0), 100 mM NaCl] for 10 min with constant shaking at 4°C. The reactions were then supplied with a ROS reaction mixture to detect their luminescence. Values are means ± sd (n = 6). **Significant differences at P < 0.01 (Student’s t test). RLU, relative light units.
(E) Rice suspension cells overexpressing OsTPR1 display enhanced chitin-triggered ROS bursts in the presence of MoChia1. Rice cells sub-cultured more than 20 times were used for the assays. Values are means ± sd (n = 6). **Significant differences at P < 0.01 (Student’s t test). RLU, relative light units.
To further verify the competition between OsTPR1 and chitin for MoChia1 binding, we used microscale thermophoresis (MST) assays to study their interactions. The results show that the OsTPR1 and MoChia1 interaction has a smaller Kd (dissociation constant; Kd = 0.052) than that of MoChia1 and chitin (Kd = 0.31), suggesting that OsTPR1 has a higher binding affinity for MoChia1 than does chitin (Figures 6B and 6C). By contrast, OsTPR1 was unable to bind with chitin (Figure 6C). In the presence of OsTPR1, chitin cannot efficiently bind to MoChia1; we found that 20 μM recombinant OsTPR1 caused the disassociation of chitin and MoChia1 (Kd = 1.34), whereas 100 μM OsTPR1 almost completely prevented the interaction of chitin and MoChia1 (Figure 6C). These data confirm the competition between OsTPR1 and chitin for MoChia1 binding, which essentially releases chitin to activate the immune response.
Because chitin binds to MoChia1, we therefore checked whether MoChia1 suppresses the chitin-triggered ROS burst as Slp1 does (Mentlak et al., 2012). Purified MoChia1 protein was found to substantially suppress the chitin-triggered ROS burst in rice cells (Figure 6D). MoChia1 is a PAMP, but it did not trigger an enhanced immune response in the presence of chitin revealed by either MAPK activation or callose deposition on the rice cell walls (Supplemental Figure 10). This result suggested that MoChia1 suppressed the chitin-triggered immune response in planta (Supplemental Figure 10). OsTPR1 significantly reverses the chitin-triggered ROS burst suppressed by MoChia1; however, OsTPR1 alone cannot induce a ROS burst (Figure 6D), implying that OsTPR1 may compete with chitin to bind to the same region of MoChia1. This competition should lead to increased levels of free chitin in the apoplast, which is perceived by the plant PRRs. To confirm this observation, we also tested the ROS burst in a suspension of rice cells overexpressing OsTPR1. MoChia1 suppresses the chitin-induced ROS burst in wild-type cells, but could not suppress the ROS burst in OsTPR1-overexpressing cells, demonstrating that OsTPR1 prevents the MoChia1-mediated suppression of the chitin-induced ROS burst in vivo (Figure 6E). Notably, OsTPR1 does not interfere with the MoChia1-activated immune response (Supplemental Figure 11).
OsTPR1 Promotes Blast Disease Resistance
TPR proteins have previously been reported to be recruited by various pathogens to enhance their virulence; however, the function of these proteins in higher plants remains elusive (Cerveny et al., 2013). Because OsTPR1 disarms the MoChia1-mediated suppression of the chitin-triggered immune response (Figures 6D and 6E), it is speculated that OsTPR1 may contribute to disease resistance in plants.
We examined OsTPR1 expression in rice leaves during blast infection, and found that the expression was significantly elevated following their inoculation with M. oryzae, implying that OsTPR1 expression is induced by infection (Figure 7A). Interestingly, we also found that MoChia1 could slightly activate OsTPR1 transcription (Figure 7B). We then generated OsTPR1 overexpression (OsTPR1-OE) and RNA interference (RNAi) lines (OsTPR1-RNAi) and confirmed the OsTPR1 expression levels using reverse transcription-quantitative PCR (RT-qPCR; Figures 7C and 7D). Then, the homozygous OsTPR1-OE plants and silenced OsTPR1-RNAi lines were used for the pathogen inoculation assays. OsTPR1-OE plants exhibited enhanced disease resistance to M. oryzae, whereas the OsTPR1-RNAi plants were more susceptible than the wild type (Figures 7E and 7F). This result is further supported by the presence of higher levels of H2O2 in the OsTPR1-OE plants following pathogen infection as revealed by DAB staining, but very low levels in the OsTPR1-RNAi plants (Figure 7G). These data indicate that OsTPR1 positively contributes to disease resistance to M. oryzae by enhancing rice immunity.
OsTPR1 Positively Contributes to Blast Disease Resistance.
(A) The OsTPR1 expression is induced by M. oryzae infection. Two-week-old rice seedlings were spray-inoculated with a conidial suspension at a concentration of 1 × 105 conidia per mL in 0.2% Tween 20. The expression of OsTPR1 was examined using RT-qPCR at the indicated times. Values are means ± sd (n = 4). **Significant differences from 0 hpi at P < 0.01 (Student’s t test).
(B) The OsTPR1 expression in DEX:MoChia1 transgenic plants. Two-week-old DEX:MoChia1 plants were treated with 30 μM DEX applied to the roots. At 24 h, the leaves were sampled and the expression of OsTPR1 was examined using RT-qPCR. Values are means ± sd (n = 4). **Significant differences from wild type at P < 0.01 (Student’s t test).
(C) and (D) OsTPR1 expression levels in the OsTPR1 transgenic plants. The leaves were sampled from 2-week-old rice seedlings of the OsTPR1-overexpressing lines and the RNAi-silenced lines. OsTPR1-OE-3 and OsTPR1-OE-11 are two independent overexpression lines. OsTPR1-RNAi-5 and OsTPR1-RNAi-7 are two independent silenced lines. Others are as in (B).
(E) OsTPR1 positively contributes to blast disease resistance in rice. The OsTPR1 overexpression (OE) lines 3 and 11 and the OsTPR1-silencing lines 5 and 7 were spray-inoculated with M. oryzae spores at a concentration of 1 × 105 conidia per mL in 0.2% Tween 20. At 5 dpi, the disease lesions were photographed. The experiment was repeated at least three times.
(F) Relative fungal biomass in (E). Values are means ± sd (n = 4). **Significant differences from wild type at P < 0.01 (Student’s t test).
(G) DAB staining of H2O2 production in OsTPR1 transgenic plants after pathogen infection. Two-week-old rice seedling leaf sheaths were inoculated with conidial spores at a concentration of 1 × 105 conidia per mL. The infected leaf tissue was stained with DAB, and the images were taken at 24 and 48 hpi.
DISCUSSION
Plant apoplast immunity is emerging as an important branch of plant innate immunity (Doehlemann and Hemetsberger, 2013). Many phytopathogens secrete virulence factors into the plant apoplast, to either suppress the perception of PAMPs by the PRRs or to directly damage the host cell structure for their own use (Jashni et al., 2015; Langner and Göhre, 2016; Ma et al., 2017). Plants are also known to secrete proteases or protease inhibitors into the apoplast during pathogen invasion, presumably targeting the virulence factors to repress infection (Doehlemann and Hemetsberger, 2013), demonstrating the importance of apoplast immunity.
As a key constituent of the fungal cell wall, chitin is a major immune activator in plant–fungi interactions. Pathogens have developed multiple strategies to sequester chitin oligomers and prevent their recognition by plant PRRs; for example, the effector protein Ecp6 secreted by C. fulvum can bind chitin and hide it from plant receptors, preventing the activation of the immune response (de Jonge et al., 2010). Another C. fulvum effector, Avr4, directly protects fungal chitin from degradation by plant chitinases (van den Burg et al., 2006). Similarly, M. oryzae secretes the effector Slp1, which competes with OsCEBiP to bind chitin oligomers, thus blocking the OsCEBiP-OsCERK1 activated immune response in rice (Mentlak et al., 2012). It is noteworthy that chitin is dynamically modified by chitinase(s) during fungi growth and infection, but their roles in plant immune responses are largely unknown. In an attempt to screen new elicitors/PAMPs during blast infection, we identified a M. oryzae chitinase, MoChia1, which plays dual roles in rice immunity (Figure 8).
Proposed Working Model.
During M. oryzae infection, the rice PRR receptor OsCERK1 recognizes fungal chitin and mounts an immune response; however, the fungus-secreted chitinase MoChia1 is able to bind chitin and repress the chitin-mediated activation of the immune response. MoChia1 is perceived by an unknown PRR, which also triggers the ROS burst. In addition, the rice plant deploys OsTPR1 to compete with chitin and bind to MoChia1, freeing chitin and thereby re-establishing the activation of the immune response.
Chitinases are widely distributed in prokaryotic and eukaryotic cells, and have diverse biological functions (Langner and Göhre, 2016); for example, the bacterial pathogen Xylella fastidiosa secretes chitinase that hydrolyzes the chitin of its host fungi and insects for use as a carbon source (Labroussaa et al., 2017). Fungal pathogens use chitinases to continuously remodel their cell wall plasticity during growth and infection (Langner and Göhre, 2016). Langner et al. (2015) also showed that chitinases were essential for cell separation in U. maydis. Because chitin is the key component of fungal cell walls, plants secrete chitinases to directly degrade the cell walls of invading fungal pathogens and halt their infection (Cletus et al., 2013). Overexpressing plant chitinases has been shown to effectively enhance the resistance of wheat (Triticum aestivum) to Fusarium pathogens and tomato (Solanum lycopersicum) to Botrytis cinerea (Cletus et al., 2013). We show here that MoChia1 is indispensable for the growth and virulence of this fungus; knocking out MoChia1 results in a faster growth rate, the production of fewer conidia, and the redistribution of chitin in the cell walls (Figure 2). MoChia1 also modulates the cell wall composition and the cell wall stress response in M. oryzae (Figure 2). In addition, knocking out MoChia1 significantly reduces the virulence of M. oryzae, because the mutant formed its appressoria more slowly than the wild-type strain (Figure 3). The higher chitin content in the fungal cell wall could therefore be responsible for the activation of the immune response and the compromised virulence of the ΔMoChia1 mutant.
Rice plants can recognize MoChia1 and mount an immune response; we demonstrate that MoChia1 exhibits the typical features of a PAMP, activating the ROS burst, causing callose deposition, and activating MAPK signaling (Figure 1). Notably, plant chitinases putatively orthologous to MoChia1 cannot activate ROS bursts in rice cells, although they possess the enzymatic activity to hydrolyze chitin (Supplemental Figure 5). Our phylogenetic analysis shows the evolutionary distance between MoChia1 and the rice chitinases; the plant chitinases are distantly related to the microbial chitinases (Supplemental Figure 2). Because rice chitinases cannot activate the ROS burst but possess chitinase activity (Supplemental Figure 5), we hypothesize that the evolution of the plant chitinases followed a different path to the fungi following the divergence of these species. The evolutionary consequence is that plants are able to carefully avoid activating their own immune systems. It is noteworthy that the MoChia1-triggered ROS burst is slower than those mediated by chitin or other PAMPs in rice (Figure 1A), implying the existence of an unknown PRR that specifically recognizes fungus-derived chitinases.
Unexpectedly, we also found that MoChia1 suppressed the chitin-triggered ROS burst in rice cells (Figure 6D). MoChia1 likely binds to chitin to prevent it from being recognized by OsCEBiP and OsCERK1, functioning in a similar manner to Slp1 during M. oryzae infection (Mentlak et al., 2012); however, we found that the rice protein OsTPR1 could disarm the MoChia1-mediated suppression of the ROS burst. The TPR family proteins contain three or more TPR structural motifs, originally identified in yeast (Sikorski et al., 1990; Cerveny et al., 2013). They usually mediate protein–protein interactions or the assembly of multi-protein complexes (Cerveny et al., 2013). These TPR proteins therefore function in a variety of cell processes, including being directly involved in virulence-associated functions in bacteria (Cerveny et al., 2013). The influenza virus was reported to recruit a 58-kD TPR protein to downregulate the interferon-induced double-stranded RNA-activated protein kinase in bovine kidney cells to avoid the kinase’s deleterious effects on viral protein synthesis and replication (Lee et al., 1994). However, the functions of the TPR proteins are not yet fully understood in higher plants. Similar to OsTPR1, several TPR family proteins were found to be located to the plasma membrane, although they have no predicted transmembrane domain (Lin et al., 2008, 2009). Our data indicate that OsTPR1 may carry atypical transmembrane domain/region (Supplemental Figure 9). In addition, the TPR domain has been found to function in protein translocation across membranes (Schlegel et al., 2007). Lin et al. reported that the TPR1 proteins in Arabidopsis and tomato interact with ethylene receptors to influence the ethylene signaling pathway, and that overexpression of the TPR1 proteins resulted in dwarf phenotypes in plants (Lin et al., 2008, 2009). However, the molecular mechanism behind these responses remains unclear. In our study, we speculate that the site at which OsTPR1 interacts with MoChia1 is probably also the MoChia1 binding site for chitin (Figure 6). The competition of OsTPR1 for MoChia1 binding essentially exposes chitin to OsCEBiP and OsCERK1, which subsequently re-activates rice immunity. As a result, transgenic rice plants overexpressing OsTPR1 display enhanced blast disease resistance (Figure 7). Although the roles of OsTPR1 need further investigation, we conclude that it functionally acts as a “decoy” chitin in this case (for model, see Figure 8); therefore, our study demonstrates that the apoplast decoy strategy also exists in higher plant.
In summary, we have identified a chitinase in M. oryzae, MoChia1, which contributes to fungal virulence by modulating fungal growth and suppressing the chitin-mediated ROS burst in the host plant. In addition, MoChia1 also acts as a PAMP. We propose a scenario that during M. oryzae infection, MoChia1 is secreted, which modulates the fungal cell wall and also binds chitin to suppress rice immune response; rice plants, however, not only deploy OsTPR1 to counteract the immune suppression caused by MoChia1, but also use the unknown PRR to perceive MoChia1 (Figure 8). This work unravels a mechanism involving the chitin-related immune response during rice blast infection. Nevertheless, the ultimate immune output should depend on the OsTPR1 and MoChia1 recognition receptor in different rice species. Our work demonstrates the complex arms race that occurs in plant apoplast immunity. Although the importance of MoChia1 in triggering the immune response of different rice varieties requires further investigation, the discovery of chitinase in the plant immune response may be useful for rice breeding. Future work should focus on the signaling components involved in MoChia1 recognition in rice.
METHODS
Plant and Fungal Strain Growth Conditions
The Magnaporthe oryzae strain Guy11 was cultured at 28°C on oatmeal agar medium (oatmeal 40 g/L, calcium carbonate 0.6 g/L, agar 30 g/L) for 2 weeks. Conidial formations were induced under white light (20,000 lux) for 2 to ∼3 days after removing the surface mycelium. Then, the spores were collected in 10 mL sterile water. Rice (Oryza sativa, subsp. japonica cv Nipponbare) plants were grown at 28°C under 16-h light and 8-h dark condition.
Preparation and Purification of Secreted Proteins from M. oryzae
To screen the elicitors, 40 L of M. oryzae growth culture was collected and spun down at 12,000 g for 10 min at 4°C. The supernatant was filtered through a 0.22 μm Millex Syringe Filter Unit (Millipore). Then, 70 g (NH4)2SO4 powder was added to 100 mL of supernatant to precipitate total proteins at 4°C for 12 h. The sediment was collected through centrifugation at 12,000 g for 10 min at 4°C. The protein pellet was resuspended and dissolved in TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 7.5)]. The proteins were loaded onto a Superdex200 10/300GL (GE Healthcare) column and subjected to fast protein liquid chromatography separation. The fractions were collected according to the absorbance peaks at 280 nm. The selected fractions were further purified by anion-exchange chromatography on Hi Trap TM QXL (GE Healthcare). The fractions that can trigger ROS burst in rice suspension cells were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS, Thermo Fisher Scientific).
Rice Leaf and Leaf Sheath Inoculation Assays
Rice leaf and leaf sheath inoculation assays followed the method used by Yang et al. (2017). For leaf inoculation, 2-week-old rice seedlings were sprayed with M. oryzae conidial suspensions at a concentration of 105 conidia/ml in 0.2% Tween-20. Inoculated plants were placed in a growth chamber at 28°C for 24 h in the dark, and switched back to the normal growth condition of a photoperiod of 16-h light (20,000 lux white fluorescent light) and 8-h dark. Photographs were taken at 5 to7 d after inoculation. The respective fungal biomass was examined by qPCR using specific primers for Pot2 of M. oryzae and normalized to the reference gene OsUbi1. SYBR Premix Taq (Toyobo) was used for qPCR. Reactions were detected by the Bio-Rad system. For rice sheath inoculation assays, the conidial suspensions were injected into the inner leaf sheaths with a conidial concentration of 105 mL by a syringe, and then the inoculated leaf sheaths were incubated under the same conditions as those that were spray inoculated. At the indicated time points, the leaf sheaths were examined under a microscope (Olympus BX51) to observe the fungal development in rice tissues.
Plant Transformation
A full-length OsTPR1 CDS was cloned from the rice variety Nipponbare, and the fragment was ligated to the binary vector pCAMBIA 1390U at sites that were digested by KpnI and BamHI to generate the overexpression construct. For the RNAi vector construction, a unique length of 231 bp to 428 bp of OsTPR1 was cloned into the RNAi vector pTCK303 (BamHI/KpnI-digested for the forward fragment and SpeI/SacI-digested for the converse fragment). The primers used are listed in Supplemental Table 2. The constructs were introduced into Nipponbare through an Agrobacterium-mediated transformation method described previously (Xiong et al., 2017).
Fungal Transformation
MoChia1, MoChia1NSP, MoChia1E137Q overexpression, and MoChia1 knockout and complementation constructs were generated as described by Li et al. (2017). Briefly, the CDS of MoChia1, MoChia1NSP, and MoChia1E137Q amplified from M. oryzae were ligated with the overexpression vector pRTN-eGFP that was digested with EcoRI and BamHI. For the knockout construct, two 800-bp flanking sequences from the upstream and downstream of the MoChia1 gene were introduced into the vector pKOV21 (EcoRI/BamHI-digested for the upstream and HindIII-digested for the downstream). For the complementation construct, a 1.1 kb fragment upstream of start codon of MoChia1 and the CDS of MoChia1 was introduced into the pRTN vector (KpnI/NotI-digested). All the ligations were performed using a One-step Cloning Kit (Vazyme Biotech, C112). The primers used are listed in Supplemental Table 2. The respective restriction enzyme digestion sites were highlighted in the primer sequences. All the constructs were confirmed by sequencing, and subsequently were transformed into protoplasts of the Guy11 or ∆MoChia1 mutant strain.
Callose Deposition and ROS Burst Assay
The leaves of 2-week-old rice seedlings that had been treated with MBP (Mock treatment), MoChia1, and Chitin (hexa-N-acetylchitohexaose, Seikagaku) were fixed in ethanol:acetic acid (3:1 [v/v]) solution for 5 h with frequently changed fresh solution. Then rice leaves were rehydrated in 70% ethanol for 2 h and 50% ethanol for 2 h, and then were kept in water overnight. After being washed with water three times, the leaves were treated with 10% sodium hydroxide (NaOH) for 1 h to make the tissues transparent. After being washed with water four times, the leaves were incubated in staining solution [150 mM K2HPO4 (pH 9.5), 0.01% aniline blue] for 4 h on an end-over-end shaker. The leaves were then observed using a Leica microscope under UV light (340 to 380 nm; Olympus BX51).
For ROS burst assays, reaction mixtures (50 mM K3PO4, 17 mM luminol, 10 mg/ml horseradish peroxidase, and 8 nM elicitor) were mixed with the rice suspension cells. Luminescence was measured continuously over a 120-min period with 1-min intervals by a Centro XS3 LB 960 Luminometer (Berthold Technologies). At least three replications were performed for all the experiments.
DAB and Mycelium Calcofluor White Staining
DAB (Sigma-Aldrich) was used for H2O2 staining according to a previously described method (Yang et al., 2017). The rice tissues were stained in 50 mL DAB solution (50 mg DAB, 0.5mM NaH2PO4, 20 μL Tween 20) and vacuumed for 5 to 10 min to eliminate air in plant tissues. Then, the tissues were continuously stained with DAB solution for 8 h on a low speed shaker. Then, the tissues were de-stained in a solution containing ethanol: lactic acid: glycerol (3:1:1) for 8 h with frequent changes of fresh solution. The tissue was observed under a light microscope (Zeiss).
Calcofluor White staining of mycelia cell wall components (chitin/cellulose) was performed using Fluorescent Brightener 28 according to the manufacturer’s protocol (10 μg/ml, Sigma-Aldrich). The fungal strains were transferred onto cover slips that contained a thin layer of complete agar medium, and cultured for 24 h. Agar pieces with mycelia were removed and stained with 10 μg/ml Calcofluor White for 10 min in darkness. The mycelia were rinsed twice with 10 N NaOH, and then were further washed three times with distilled, deionized water and viewed under a fluorescence microscope (Nikon).
Measurement of Chitin Contents in M. oryzae
The chitin content was determined by measuring the amount of glucosamine released by acid hydrolysis from fungal cell walls, according to a previously described method (Guerriero et al., 2010). One gram of freshly harvested mycelia or conidia was ground in liquid nitrogen and then homogenized in 5 mL of deionized water. After centrifugation at 13,000 g for 10 min at 4°C, the pellets were lyophilized overnight (Labconco). For each 5 mg of the dried pellets, 1 mL of 6 M HCl was added. After the samples were hydrolyzed at 100°C for 4 h, the hydrolysis was adjusted with 10 N NaOH to pH 7.0. An aliquot (0.2 mL) of the resulting mixture was added to 0.25 mL of 4% acetyl acetone in 1.25 M sodium carbonate and heated for 30 min at 100°C. After cooling down, 2 mL ethanol and 0.25 mL of Ehrlich reagent (1.6 g of N,N-dimethyl-p-aminobenzaldehyde in 60 mL of a 1:1 mixture of ethanol and concentrated HCl) was added to the mixture, and the mixture was heated for 1 h at 60°C. The mixture was further centrifuged at 13,000 g for 10 min at room temperature, and the supernatant was measured for absorbance at 530 nm. The chitin content was calculated based on the standard curve established by measuring the absorbance of known amounts of glucosamine hydrochloride (Sigma, G4875).
Chitinase Activity Assay
Colloidal chitin was prepared according to a previously described method (Niu et al., 2016). Briefly, 10 g chitin power was ground in 40 mL acetone, and then 400 mL concentrated HCl was added in the homogenate. The mixture was kept at 4°C for 24 h and then filtered through glass wool to 2 L of 50% prechilled ethanol with constant mixing. After centrifugation at 10,000 rpm for 20 min at 4°C, the white precipitate was washed in cold prechilled distilled water several times until the pH was close to 5.5. The supernatant was discarded, and 1 L of distilled water was added to form 0.5% colloidal chitin. Chitinase activity was measured using colloidal chitin as the substrate. One microgram of recombinant protein was incubated with 0.25 mg colloidal chitin at 37°C for 1 h in 100 μL 50 mM sodium acetate buffer (pH 7.0). The reaction was terminated by adding 100 μL 3,5-dinitrosalicylic acid (DNS) following 5 min heating at 100°C. After cooled in an ice bath, the mixture was diluted with 800 μL H2O. The undigested chitin was removed by centrifugation. The absorbance of supernatant was monitored at OD 565 nm. The standard curve was generated by the reaction of GlcNAc and DNS. One unit of chitinase activity was defined as the amount of enzyme required to produce 1 μmol of GlcNAc/h under the above conditions.
Rice Apoplastic Protein Extraction
The rice apoplast protein extraction was prepared as described (Kim et al., 2013) with slight modification. Briefly, the leaves were washed with distilled, deionized water three times to remove any dust. Then, the leaves were vacuum infiltrated with distilled, deionized H2O2 under 7.5 psi for 5 min. Then, the leaves were loaded into 50 mL centrifuge tubes and centrifuged at 1000 g for 5 min. The extracted apoplastic proteins were further concentrated in the Millipore tubes by the TCA-DOC method (Haslam et al., 2003; O’Leary et al., 2014), and the proteins were resuspended in lamelli buffer. The proteins were separated on a SDS-PAGE gel and probed by the FLAG (Sigma, F3165) and ribulose-1,5-bis-phosphate carboxylase/oxygenase (Huaxingbio, HX1989) antibodies, respectively.
Split-Luciferase Complementation Assay
The split-luciferase complementation assay was performed as described (Luo et al., 2017). Agrobacterium tumefaciens (strain C58C1) carrying the indicated nLUC and cLUC constructs was mixed and infiltrated into the leaves of 4-week-old N. benthamiana plants using a 1-mL needleless syringe. Two days after infiltration, the leaves were rubbed with 0.5 mM luciferin and kept in the dark for 5 min to quench the fluorescence. A cooled CCD imaging apparatus (Roper Scientific) was used to capture luciferase images.
BiFC Assay
The pSAT1-nYFP and pSAT1-cYFP plasmids were used for the BiFC assay. The CDS of MoChia1 and MoChia1NSP were cloned into pSAT1-cYFP at EcoRI/SmaI sites, and the CDS of OsTPR1 was cloned into pSAT1-nYFP at SalI/BamHI sites. The Agrobacterium tumefaciens strain C58C1 carrying the indicated constructs was mixed and infiltrated into the leaves of 4-week-old N. benthamiana plants. Infiltrated leaves were observed 36 to 48 h later using a confocal laser scanning microscope (Leica Model TCS SP8).
Subcellular Localization
MoChia1, OsTPR1, and GhPOD10 proteins with GFP-tag at the C-terminal were co-expressed with PCD1002-CFP, respectively, in N. benthamiana. Forty-eight hours later, the expressed proteins were observed using a confocal laser microscope (Leica Model TCS SP8). Rice protoplast preparation and plasmid transformation were performed according to the methods of Xiong et al. (2017). The excitation wavelengths and emission filters are as follows: 488 nm/band-pass 500 to 550 nm for GFP, and 433 nm/band-pass 475 to 503 nm for CFP. Confocal images were analyzed using Leica LAS AF software.
The OsTPR1 extracellular domain/region was identified by protease protection assays using trypsin (Amresco) or protease K (Sigma). The proteases were dissolved in 50 mM Tris-HCl, pH 8.0. The rice protoplast or N. benthamiana that expressed OsTPR1-GFP or GFP proteins were incubated with proteases at 28°C. The anti-GFP antibody (TransGen Biotech, HT801) was used to examine the protein degradation.
GST and Chitin Pull-Down Assays
The MoChia1, MoChia1E37Q, MoChia1CBD, and OsTPR1 were cloned into respective pGEX-4T-1 and/or pMAl-C4X vectors using a One-step Cloning Kit (Vazyme Biotech, C112) and expressed in E. coli strain BL21 to produce recombinant proteins. The GST pull-down assay was performed using the method described by Luo et al. (2017). The chitin pull-down assay was performed using the method described by Liu et al. (2012). Briefly, insoluble chitin was incubated with purified recombinant MBP-OsTPR1 in binding buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100] at 4°C for 2 h with constant shaking. The glycan was spun down by centrifugation at 1000 g at 4°C for 3 min. The pellet was washed five times with washing buffer [20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.2% Nonidet P-40] and then boiled with SDS-PAGE loading buffer. The chitin-associated MBP-MoChia1 was detected by immunoblotting using anti-MBP antibody (TransGen Biotech, HT701).
MST Analysis
Binding reactions of recombinant MBP-MoChia1 to MBP-OsTPR1 or chitin was measured by MST in a Monolith NT.Label Free (Nano Temper Technologies GMBH) instrument that detects changes in size, charge, and conformation induced by binding. Labeled MBP-MoChia1 (10 μM) was displaced by a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, and 0.05% (v/v) Tween 20. A range of concentrations of MBP-OsTPR1 or chitin in the assay buffer [50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 10 mM MgCl2, 0.05% Tween 20] was incubated with labeled protein (1:1, v/v) for 10 min. The sample was loaded into the NT.Label Free standard capillaries and measured with 20% LED power and 80% MST power. The KD Fit function of the Nano Temper Analysis Software (Version 1.5.41) was used to fit the curve and calculate the value of the dissociation constant (Kd).
Phylogenetic Analyses
The amino acid sequences of the chitinase proteins were downloaded from the UniProt website (https://www.uniprot.org/). Sequence alignment was performed with ClustalW (Supplemental Data Set). A neighbor-joining method implemented in MEGA7.0 was used to generate the phylogenetic tree. The bootstrap values indicated at the nodes in the phylogenetic tree are based on 1000 replications.
Statistical Analysis
All the data were analyzed using a one-way ANOVA or two-tailed Student’s t test with SPSS 18.0. The values represented as means ± sd.
Accession Numbers
Sequence data from this article can be found in the GenBank database libraries under the following accession numbers: MoChia1, MGG_08054; 4HNR, MGG_07216; AIB1, MGG_07219; BUF1, MGG_02252; RSY1, MGG_05059; MoActin, MGG_03982; OsTPR1, LOC_Os10g34540; two rice chitinase genes, LOC_Os04g30770 and LOC_Os05g33130; OsRbohA, LOC_Os01g53294; OsRbohB, LOC_Os09g26660; OsRbohD, LOC_Os05g38980; OsPR10, LOC_Os03g18850; OsActin, LOC_Os10g36650.
Supplemental Data
Supplemental Figure 1. The identification of MoChia1 in M. oryzae.
Supplemental Figure 2. Phylogenetic tree of chitinase gene families.
Supplemental Figure 3. MoChia1 is a secreted protein.
Supplemental Figure 4. MoChia1 chitinase activity is not essential for immune activation.
Supplemental Figure 5. Rice chitinases possess enzymatic activities but do not cause a ROS burst in rice suspension cells.
Supplemental Figure 6. Strategy for the targeted gene knockout of MoChia1.
Supplemental Figure 7. The phenotype of MoChia1-overexpressing strains.
Supplemental Figure 8. △MoChia1 activates stronger immune responses than the WT in rice.
Supplemental Figure 9. MoChia1 interacts with the C-terminal of OsTPR1.
Supplemental Figure 10. Chitin and MoChia1 activated immune responses in rice.
Supplemental Figure 11. Comparison of ROS burst activated by MoChia1 in rice suspension cells of WT and OsTPR1-OE.
Supplemental Table 1. The proteins identified by LC-MS/MS.
Supplemental Table 2. Primers used in this study.
Supplemental Data Set. Text file of alignment corresponding to the phylogenetic analysis in Supplemental Figure 2.
Dive Curated Terms
The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:
Acknowledgments
We thank Prof. Youliang Peng for kindly providing the pRTN-eGFP and pKOV21 plasmids and Prof. Junfeng Liu at Chinese Agricultural University for bioinformatics assays. We also thank Yao Wu for technical assistance on the use of MST. The work was supported by the Chinese Academy of Sciences (CAS) (Strategic Priority Research Program Grant XDB11020300), the National Natural Science Foundation of China (NSFC) (31570252, 31601629), the start-up fund of ‘One Hundred Talents’ program of the Chinese Academy of Sciences, and by the grants from the State Key Laboratory of Plant Genomics (Grant O8KF021011 to J.L.).
AUTHOR CONTRIBUTIONS
C.Y. and J.L. conceived and designed the experiments, and wrote the article; C.Y., Y.Y., J.H., and F.M. performed most of the experiments; J.P. and Y.T. generated the transgenic plants; Q.Z., A.I., and N.X. helped with the data analysis.
Footnotes
↵1 These authors contributed equally to this work.
- Received May 14, 2018.
- Revised November 19, 2018.
- Accepted December 31, 2018.
- Published January 4, 2019.
References
