Fit accuracy of resin crown on a dental model fabricated using fused deposition modeling 3D printing and a polylactic acid filament.

Purpose We considered the possibility of reducing industrial waste by fabricating and reusing dental models prepared using a fused deposition modeling (FDM) 3D printer and polylactic acid (PLA) filaments. The purpose of this study was to verify the accuracy of models fabricated using FDM and PLA.Methods The same provisional crown was used to check the marginal fit on PLA models prepared using an intraoral scanner (IOS) and FDM, plaster models made with silicone impression material and plaster, and resin models prepared using an IOS and stereolithography apparatus (SLA) 3D printer. The marginal fit was measured using micro-computed tomography at four points on the tooth: the buccal center (B), palatal center (P), mesial center (M), and distal center (D) points.Results At point B, the marginal gaps were 118 ± 21.7, 62 ± 16.4, and 50 ± 26.5 µm for the PLA, resin, and plaster models, respectively, with a significant difference between the PLA model and the other two. However, the marginal gap at all other measurement points was not significantly different between the models (P > 0.05).Conclusions We compared the accuracy of the models fabricated using the FDM, SLA, and conventional methods. The combination of FDM and PLA filaments showed no significant differences from the other models, except at point B, indicating its usefulness. Therefore, FDM and PLA may become necessary materials for dental treatment in the future.

Transcriptome analysis of tomato plants following salicylic acid-induced immunity against Clavibacter michiganensis ssp. michiganensis.

Salicylic acid (SA) is known to be involved in the immunity against Clavibacter michiganensis ssp. michiganensis (Cmm) that causes bacterial canker in tomato. To identify the candidate genes associated with SA-inducible Cmm resistance, transcriptome analysis was conducted via RNA sequencing in tomato plants treated with SA. SA treatment upregulated various defense-associated genes, such as PR and GST genes, in tomato cotyledons. A comparison of SA- and Cmm-responsive genes revealed that both SA treatment and Cmm infection commonly upregulated a large number of genes. Gene Ontology (GO) analysis indicated that the GO terms associated with plant immunity were over-represented in both SA- and Cmm-induced genes. The genes commonly downregulated by both SA treatment and Cmm infection were associated with the cell cycle and may be involved in growth and immunity trade-off through cell division. After SA treatment, several proteins that were predicted to play a role in immune signaling, such as resistance gene analogs, Ca2+ sensors, and WRKY transcription factors, were transcriptionally upregulated. The W-box element, which was targeted by WRKYs, was over-represented in the promoter regions of genes upregulated by both SA treatment and Cmm infection, supporting the speculation that WRKYs are important for the SA-mediated immunity against Cmm. Prediction of protein-protein interactions suggested that genes encoding receptor-like kinases and EF-hand proteins play an important role in immune signaling. Thus, various candidate genes involved in SA-inducible Cmm resistance were identified.

Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity.

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction.

A Seed-Borne Bacterium of Rice, Pantoea dispersa BB1, Protects Rice from the Seedling Rot Caused by the Bacterial Pathogen Burkholderia glumae.

Seedling rot, caused by the bacterial pathogen Burkholderia glumae, is a major disease of rice. It originates from pathogen-contaminated seeds and is thus mainly controlled by pesticide treatments of seeds. We previously demonstrated that the seed-borne bacteria of rice may be a useful and sustainable alternative to pesticides to manage seedling rot, but they are limited in terms of variety. Here, we report that another seed-borne bacterium, Pantoea dispersa BB1, protects rice from B. glumae. We screened 72 bacterial isolates from rice seeds of three genetically different cultivars inoculated or non-inoculated with B. glumae. 16S rRNA gene sequencing revealed that pathogen inoculation affected the composition of culturable seed-borne bacterial communities and increased the presence of Pantoea and Paenibacillus species. Among three Pantoea and Paenibacillus isolates that exhibit tolerance to toxoflavin, a virulence factor of B. glumae, P. dispersa BB1 significantly mitigated the symptoms of rice seedling rot. The culture filtrate of BB1 inhibited the growth of B. glumae in vitro, suggesting that this isolate secretes antibacterial compounds. Seed treatment with BB1 suppressed pathogen propagation in plants, although seed treatment with the culture filtrate did not. Because BB1 did not show pathogenicity in rice, our findings demonstrate that BB1 is a promising biocontrol agent against seedling rot.

Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-22 and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

Surveillance of Pathogenicity of Rhizoctonia solani Japanese Isolates with Varied Anastomosis Groups and Subgroups on Arabidopsis thaliana.

Rhizoctonia solani is a necrotrophic plant pathogen with a wide host range. R. solani is a species complex consisting of thirteen anastomosis groups (AGs) defined by compatibility of hyphal fusion reaction and subgroups based on cultural morphology. The relationship between such classifications and host specificity remains elusive. Here, we investigated the pathogenicity of seventeen R. solani isolates (AG-1 to 7) in Japan towards Arabidopsis thaliana using leaf and soil inoculations. The tested AGs, except AG-3 and AG-6, induced symptoms in both methods with variations in pathogenicity. The virulence levels differed even within the same AG and subgroup. Some isolates showed tissue-specific infection behavior. Thus, the AGs and their subgroups are suggested to be not enough to define the virulence (host and tissue specificity) of R. solani. We also evaluated the virulence of the isolates on Arabidopsis plants pretreated with salicylic acid, jasmonic acid, and ethylene. No obvious effects were detected on the symptom formation by the virulence isolates, but ethylene and salicylic acid slightly enhanced the susceptibility to the weak and nonvirulent isolates. R. solani seems to be able to overcome the induced defense by these phytohormones in the infection to Arabidopsis.

Transcriptome analysis of Clavibacter michiganensis subsp. michiganensis-infected tomatoes: a role of salicylic acid in the host response.

Bacterial canker of tomato (Solanum lycopersicon) caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is an economically important disease. To understand the host defense response to Cmm infection, transcriptome sequences in tomato cotyledons were analyzed by RNA-seq. Overall, 1788 and 540 genes were upregulated and downregulated upon infection, respectively. Gene Ontology enrichment analysis revealed that genes involved in the defense response, phosphorylation, and hormone signaling were over-represented by the infection. Induced expression of defense-associated genes suggested that the tomato response to Cmm showed similarities to common plant disease responses. After infection, many resistance gene analogs (RGAs) were transcriptionally upregulated, including the expressions of some receptor-like kinases (RLKs) involved in pattern-triggered immunity. The expressions of WRKYs, NACs, HSFs, and CBP60s encoding transcription factors (TFs) reported to regulate defense-associated genes were induced after infection with Cmm. Tomato genes orthologous to Arabidopsis EDS1, EDS5/SID1, and PAD4/EDS9, which are causal genes of salicylic acid (SA)-deficient mutants, were upregulated after infection with Cmm. Furthermore, Cmm infection drastically stimulated SA accumulation in tomato cotyledons. Genes involved in the phenylalanine ammonia lyase pathway were upregulated, whereas metabolic enzyme gene expression in the isochorismate synthase pathway remained unchanged. Exogenously applied SA suppressed bacterial growth and induced the expression of WRKYs, suggesting that some Cmm-responsive genes are regulated by SA signaling, and SA signaling activation should improve tomato immunity against Cmm.

Identification of effector candidate genes of Rhizoctonia solani AG-1 IA expressed during infection in Brachypodium distachyon.

Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon. R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes (RsSEPGs) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1-3 and 29 RsSEPGs in the cluster 4-6 are expected to be involved in biotrophic and necrotrophic interactions, respectively.

BdWRKY38 is required for the incompatible interaction of Brachypodium distachyon with the necrotrophic fungus Rhizoctonia solani.

Rhizoctonia solani is a soil-borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3-1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession-specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3-1 and Tek-3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY-dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling.

Parental legacy and regulatory novelty in Brachypodium diurnal transcriptomes accompanying their polyploidy.

Polyploidy is a widespread phenomenon in eukaryotes that can lead to phenotypic novelty and has important implications for evolution and diversification. The modification of phenotypes in polyploids relative to their diploid progenitors may be associated with altered gene expression. However, it is largely unknown how interactions between duplicated genes affect their diurnal expression in allopolyploid species. In this study, we explored parental legacy and hybrid novelty in the transcriptomes of an allopolyploid species and its diploid progenitors. We compared the diurnal transcriptomes of representative Brachypodium cytotypes, including the allotetraploid Brachypodium hybridum and its diploid progenitors Brachypodium distachyon and Brachypodium stacei. We also artificially induced an autotetraploid B. distachyon. We identified patterns of homoeolog expression bias (HEB) across Brachypodium cytotypes and time-dependent gain and loss of HEB in B. hybridum. Furthermore, we established that many genes with diurnal expression experienced HEB, while their expression patterns and peak times were correlated between homoeologs in B. hybridum relative to B. distachyon and B. stacei, suggesting diurnal synchronization of homoeolog expression in B. hybridum. Our findings provide insight into the parental legacy and hybrid novelty associated with polyploidy in Brachypodium, and highlight the evolutionary consequences of diurnal transcriptional regulation that accompanied allopolyploidy.

Benzothiadiazole, a plant defense inducer, negatively regulates sheath blight resistance in Brachypodium distachyon.

Plant defense inducers that mimic functions of the plant immune hormone salicylic acid (SA) often affect plant growth. Although benzothiadiazole (BTH), a synthetic analog of SA, has been widely used to protect crops from diseases by inducing plant defense responses, we recently demonstrated that SA, but not BTH, confers resistance against Rhizoctonia solani, the causal agent of sheath blight disease, in Brachypodium distachyon. Here, we demonstrated that BTH compromised the resistance of Bd3-1 and Gaz4, the two sheath blight-resistant accessions of B. distachyon, which activate SA-dependent signaling following challenge by R. solani. Moreover, upon analyzing our published RNA-seq data from B. distachyon treated with SA or BTH, we found that BTH specifically induces expression of genes related to chloroplast function and jasmonic acid (JA) signaling, suggesting that BTH attenuates R. solani resistance by perturbing growth-defense trade-offs and/or by inducing a JA response that may increase susceptibility to R. solani. Our findings demonstrated that BTH does not work as a simple mimic of SA in B. distachyon, and consequently may presumably cause unfavorable side effects through the transcriptional alteration, particularly with respect to R. solani resistance.

OsCERK1 plays a crucial role in the lipopolysaccharide-induced immune response of rice.

Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2 O2 ) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different.

Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon.

Rhizoctonia solani is a soil-borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)-dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone-induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA-deficient NahG transgenic rice and the sheath blight-resistant B. distachyon accessions, Bd3-1 and Gaz-4, which activate SA-dependent signaling on inoculation. Our findings suggest a hemi-biotrophic nature of R. solani, which can be targeted by SA-dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon.

BACKGROUND: Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium. RESULTS: We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice. CONCLUSION: We propose that the Brachypodium immune hormone marker genes identified in this study will be useful to plant pathologists who use Brachypodium as a model pathosystem, because the timing of their transcriptional activation matches that of the disease resistance response. Our results using Brachypodium also suggest that monocots share a characteristic immune system, defined as the common defence system, that is different from that of dicots.

The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice.

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

Targeted gene disruption of OsCERK1 reveals its indispensable role in chitin perception and involvement in the peptidoglycan response and immunity in rice.

OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.

CEBiP is the major chitin oligomer-binding protein in rice and plays a main role in the perception of chitin oligomers.

CEBiP, a plasma membrane-localized glycoprotein of rice, directly binds with chitin elicitors (CE), and has been identified as a receptor for CE by using CEBiP-RNAi rice cells. To further clarify the function of CEBiP, we produced CEBiP-disrupted rice plants by applying an efficient Agrobacterium-mediated gene-targeting system based on homologous recombination, which has recently been developed for rice. Homologous recombination occurred at the CEBiP locus in ~0.5 % of the positive/negative selected calli. In the self-pollinated next generation, it was confirmed that the first exon of CEBiP was replaced with the hygromycin selection cassette as designed, and that the expression of CEBiP was completely deficient in homozygous cebip lines. Affinity-labeling analysis using biotinylated N-acetylchitooctaose demonstrated that CEBiP is the major CE-binding protein in rice cultured cells and leaves, which was consistent with the result that the response to CE in cebip cells was greatly diminished. Nevertheless, we observed a significant decrease in disease resistance against Magnaporthe oryzae, the causal agent of rice blast disease, only when the cebip leaf sheaths were inoculated with a weakly virulent strain, suggesting that CE perception during the infection process of M. oryzae is limited. The response to peptidoglycan and lipopolysaccharides in cebip cells was not affected, strongly suggesting that CEBiP is a CE-specific receptor.

Expression of the chimeric receptor between the chitin elicitor receptor CEBiP and the receptor-like protein kinase Pi-d2 leads to enhanced responses to the chitin elicitor and disease resistance against Magnaporthe oryzae in rice.

We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors.

Surface α-1,3-glucan facilitates fungal stealth infection by interfering with innate immunity in plants.

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant's defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus Magnaporthe oryzae.

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.