OHTAAtsushi – Crop Evolution Lab.

2023-01-232023-01-23

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

Abstract

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 M. oryzae 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 M. oryzae 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. M. *oryzae* 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 M. *oryzae* 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 *AVR*s. 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 M. *oryzae* 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

2023-01-232023-01-23

Updated our publications list.

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

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

> Publication

2023-01-202023-01-23

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

Abstract

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 N. benthamiana 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 N. benthamiana 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 N. *benthamiana* plants (right).

2023-01-202023-01-23

Updated our publications list.

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

Kourelis, J., Contreras, M. P., Harant, A., Pai, H., Lüdke, D., Adachi, H., Derevnina, L., Wu, C.-H., & Kamoun, S. (2022). The helper NLR immune protein NRC3 mediates the hypersensitive cell death caused by the cell-surface receptor Cf-4. PLOS Genetics, 18(9), e1010414-. https://doi.org/10.1371/journal.pgen.1010414

> Publications

2023-01-192023-01-23

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

Abstract

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.