Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector

New paper from our laboratory

Sugihara, Y., Abe, Y., Takagi, H., Abe, A., Shimizu, M., Ito, K., Kanzaki, E., Oikawa, K., Kourelis, J., Langner, T., Win, J., Białas, A., Lüdke, D., Contreras, M. P., Chuma, I., Saitoh, H., Kobayashi, M., Zheng, S., Tosa, Y., Banfield, M., Kamoun, S., Terauchi, R., & Fujisaki, K. (2023). Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector. PLOS Biology, 21(1), e3001945-. https://doi.org/10.1371/journal.pbio.3001945


Studies focused solely on single organisms can fail to identify the networks underlying host–pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to Moryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the Moryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

Fig. Moryzae genetic analysis identifies an AVR gene, AVR-Mgk1, encoded on a mini-chromosome.
(A) Schematic representations of the F1 progeny generated after a cross between Moryzae isolates TH3o and O23. We subjected all F1 progeny to whole-genome sequencing. O23 possesses a mini-chromosome. (B) Genetic association of the TH3o × O23 F1 progeny using infection lesion size on RIL #58 (Pish -, Piks +) rice plants as a trait. The vertical axis indicates -log10(p), where the p-value is how likely the marker shows association with a trait due to random chance. The dashed line shows the p-value corresponding to a false discovery rate of 0.05. The association analysis based on the O23 reference genome identified AVR-Mgk1, encoded on the mini-chromosome sequence O23_contig_1, as an AVR gene. O23_contig_1 was not present in the TH3o genome and was unique to the O23 genome. We used 7,867 SNP markers for chromosomes 1–7 and 265 presence/absence markers for the other contigs. (C) p-values for O23_contig_1 with annotated AVRs. We also detected AVR-Pita and AVR-PikD in O23_contig_1. AVR-PikD in O23_contig_1 contains a frameshift mutation, so we named this variant AVR-PikD_O23. The region encoding 2 AVR-Mgk1 genes and showing lower p-values is highlighted in green. Nucleotide sequences of the 2 AVR-Mgk1 genes, arranged in a head-to-head orientation, are identical. (D) Results of punch inoculation assays using Moryzae isolate Sasa2 transformed with AVR-PikD or AVR-Mgk1. Wild-type Sasa2 infected all the cultivars tested in this study. The Sasa2 transformant expressing AVR-PikD infected RIL #58 (Piks), but that expressing AVR-Mgk1 did not infect RIL #58 (Piks) or Tsuyuake (Pikm) rice plants. (E) Quantification of the lesion size in (D). Asterisks indicate statistically significant differences (p < 0.001, two-sided Welch’s t test). The data underlying Fig 4B and 4C and 4E can be found in S1 Data. AVR, avirulence; RIL, recombinant inbred line; SNP, single nucleotide polymorphism. https://doi.org/10.1371/journal.pbio.3001945.g004

An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana

This paper has been published in which Dr. Adachi is the corresponding author.

Adachi, H., Sakai, T., Harant, A., Pai, H., Honda, K., Toghani, A., Claeys, J., Duggan, C., Bozkurt, T. O., Wu, C., & Kamoun, S. (2023). An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana. PLOS Genetics, 19(1), e1010500-. https://doi.org/10.1371/journal.pgen.1010500


The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC (NLR-REQUIRED FOR CELL DEATH) network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX in Nbenthamiana markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, spot gene silencing of NRCX in mature Nbenthamiana leaves doesn’t result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.

Author summary

Plants have an effective immune system to fight off diverse pathogens such as fungi, oomycetes, bacteria, viruses, nematodes and insects. In the first layer of their immune system, receptor proteins act to detect pathogens and activate the defense response. Plant genomes encode very large and diverse repertoires of immune receptors, some of which function in pairs or as complex receptor networks. However, the immune system can come at a cost for plants and inappropriate receptor activation results in growth suppression and autoimmunity. Here, we show that an atypical immune receptor gene functions as a modulator of the immune receptor network. This type of receptor gene evolved to maintain homeostasis of the immune system and balance fitness trade-offs between growth and immunity. Further understanding how plants regulate their immune receptor system should help guide breeding disease resistant crops with limited fitness penalties.

Fig. Modulator NLR has evolved to maintain NLR network homeostasis.

We propose that “Modulator NLR” contributes to NLR immune receptor network homeostasis during plant growth. A modulator NRCX has a similar sequence signature with helper MADA-CC-NLRs, but unlike helpers, NRCX lacks the functional MADA motif to execute cell response. NRCX modulates the NRC2/NRC3 subnetwork composed of multiple sensor NLRs and cell-surface receptor (left). Loss of function of NRCX leads to the enhanced hypersensitive response and dwarfism in Nbenthamiana plants (right).

Isolation of Pikps, an allele of Pik, from the aus rice cultivar Shoni

New paper from our laboratory

Kovi, B., Sakai, T., Abe, A., Kanzaki, E., Terauchi, R., & Shimizu, M. (2023). Isolation of Pikps, an allele of Pik, from the aus rice cultivar Shoni. Genes & Genetic Systems, advpub. https://doi.org/10.1266/ggs.22-00002


Blast disease caused by the filamentous fungus Pyricularia oryzae (syn. Magnaporthe oryzae) is one of the most destructive diseases of rice (Oryza sativa L.) around the globe. An aus cultivar, Shoni, showed resistance against at least four Japanese P. oryzae isolates. To understand Shoni’s resistance against the P. oryzae isolate Naga69-150, genetic analysis was carried out using recombinant inbred lines developed by a cross between Shoni and the japonica cultivar Hitomebore, which is susceptible to Naga69-150. The result indicated that the resistance was controlled by a single locus, which was named Pi-Shoni. A QTL analysis identified Pi-Shoni as being located in the telomeric region of chromosome 11. A candidate gene approach in the region indicated that Pi-Shoni corresponds to the previously cloned Pik locus, and we named this allele Pikps. Loss of gene function mediated by RNA interference demonstrated that a head-to-head-orientated pair of NBS-LRR receptor genes (Pikps-1 and Pikps-2) are required for the Pikps-mediated resistance. Amino acid sequence comparison showed that Pikps-1 is 99% identical to Pikp-1, while Pikps-2 is identical to Pikp-2. Pikps-1 had one amino acid substitution (Pro351Ser) in the NBS domain as compared to Pikp-1. The recognition specificity of Pikps against known AVR-Pik alleles is identical to that of Pikp.

Fig. 1 Resistance assay of 125 RILs against the Naga69-150 isolate of Pyricularia oryzae.
(A) The disease severity index (DI) was employed in evaluating phenotypes of RILs after spray inoculation of the fungus. DI = 0, no symptoms; DI = 1, 0 to 20% infected leaf area; DI = 2, over 20% infected leaf area. Representative leaves for each category are shown. Scale bars, 0.5 cm. (B) Frequency distribution of the DI for 125 RILs derived from a cross between Hitomebore and Shoni. Arrows indicate approximate value obtained for the parental (Hitomebore and Shoni) lines. The DI score of each RIL is represented by the average value of two technical replications.
Fig. 2 Identification of Pi-Shoni (Pikps) conferring resistance on Shoni against Naga69-150.
(A) QTL analysis of DI scores obtained from the 125 RILs. The dashed line indicates the significance threshold (-log10(P) > 3.36). (B) Comparative genomic mapping of the 11 NLR genes in Nipponbare within the scaffold bctg00000014 of the Shoni genome assembly. The black arrows indicate NLR genes. Pikps-1 and Pikps-2 correspond to LOC_Os11g46200 and LOC_Os11g46210, respectively

Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins

This paper has been published in which Prof. Terauchi is the corresponding author.

Takeda, T., Takahashi, M., Shimizu, M., Sugihara, Y., Yamashita, T., Saitoh, H., Fujisaki, K., Ishikawa, K., Utsushi, H., Kanzaki, E., Sakamoto, Y., Abe, A., & Terauchi, R. (2022). Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins. PLOS Pathogens18(9), e1010792-. https://doi.org/10.1371/journal.ppat.1010792


When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with Moryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against Moryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

Author summary

Plants have evolved various activity-inhibiting proteins as a defense against fungal cell wall-degrading enzymes (CWDEs), but how plants counteract the function of fungal enzymes containing carbohydrate binding modules (CBMs) remains unknown. Here, we demonstrate that OsRMC, a member of the cysteine-rich repeat secretion protein family, interacts with fungal CBM1. OsRMC binding to CBM1 of a blast fungal xylanase blocks access to cellulose, resulting in the inhibition of xylanase enzymatic activity. Our study provides significant insights into plant countermeasures against CWDEs in the apoplastic space during plant-fungal pathogen interactions. It also reveals a molecular function of the DUF26 domain widely distributed in plant proteins.

Various CBM1-containing CWDEs are secreted from Moryzae. OsRMC is secreted to apoplastic space and binds to CBM1 of the enzymes, resulting inhibition of the enzymatic activities. OsCBMIP-K binds to CBM1 of the enzymes and may potentially be involved in signal transduction. 

Activation and Regulation of NLR Immune Receptor Networks


Kourelis, J., & Adachi, H. (2022). Activation and Regulation of NLR Immune Receptor Networks. Plant and Cell Physiology, pcac116. https://doi.org/10.1093/pcp/pcac116


Plants have many types of immune receptors that recognize diverse pathogen molecules and activate the innate immune system. The intracellular immune receptor family of nucleotide-binding domain leucine-rich repeat–containing proteins (NLRs) perceive translocated pathogen effector proteins and execute a robust immune response, including programmed cell death. Many plant NLRs have functionally specialized to sense pathogen effectors (sensor NLRs) or to execute immune signalling (helper NLRs). Sub-functionalized NLRs form a network-type receptor system known as the NLR network. In this review, we highlight the concept of NLR networks, discussing how they are formed, activated, and regulated. Two main types of NLR networks have been described in plants: the ADR1/NRG1 network and the NRC network. In both networks, multiple helper NLRs function as signalling hubs for sensor NLRs and cell surface–localized immune receptors. Additionally, the networks are regulated at the transcriptional and posttranscriptional levels, as well as being modulated by other host proteins to ensure proper network activation and prevent autoimmunity. Plant pathogens in turn have converged on suppressing NLR networks, thereby facilitating infection and disease. Understanding the NLR immune system at the network level could inform future breeding programs by highlighting the appropriate genetic combinations of immunoreceptors to use while avoiding deleterious autoimmunity and suppression by pathogens.