The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding

Abstract

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

Figure 1.

AVR1-CO39 Interacts Physically with RGA5. (A) Screening of a rice yeast two-hybrid cDNA library with AVR1-CO39 identified three different clones for AVR1-CO39 Interactor 1 (ACI1-L, -M, and -S). Interactions were assayed on synthetic triple dropout (TDO) medium lacking Trp, Leu, and His and supplemented with 10 mM 3-amino-1,2,4-triazole (3AT). Autoactivation of BD:AVR1-CO39 and AD:ACI1 constructs was tested using empty pGADT7-AD (Empty-AD) and empty pGBKT7-BD (Empty-BD) vectors. Photos show yeast colonies after 4 d of growth. (B) ACI1-L, -M, and -S align to the 3′ extremity of the RGA5 gene located adjacent to the RGA4 gene on chromosome 11 (Chr. 11) of rice varieties Sasanishiki and CO39. nt, nucleotides.

Figure 2.

RGA4 and RGA5 Confer Pi-CO39 Resistance. (A) rga4 mutant lines are compromised in Pi-CO39 resistance. Transgenic M. oryzae Guy11-AVR1-CO39 or Guy11-EV (empty vector) strains were spray inoculated on 3-week-old plants of the rice cultivar Sasanishiki and Sasanishiki ethyl methanesulfonate mutant lines rga4-1493 and rga4-2127 (Okuyama et al., 2011). Development of disease and HR symptoms was followed 7 d after inoculation to determine susceptibility or resistance. Identical results were obtained in two independent inoculation experiments using the two independent mutant lines each time. Pictures show typical symptoms at 7 d after inoculation. (B) RGA4 and RGA5 are required for Pi-CO39 resistance. Transgenic rice lines of the cultivar Kanto51 (pi-CO39, pia) carrying the empty pCambia1300 binary vector (empty), a genomic RGA4 construct (RGA4), a genomic RGA5 construct (RGA5), or RGA4 and RGA5 constructs (RGA4 + RGA5) were challenged by spray inoculation with the Guy11-AVR1-CO39 or the Guy11-EV strains. Only transgenic rice lines carrying both RGA4 and RGA5 were resistant to the AVR1-CO39–expressing M. oryzae strain. Identical results were obtained in two independent inoculation experiments. Pictures show typical symptoms at 7 d after inoculation.

Figure 3.

RGA5 Is Subject to Alternative Splicing and Produces Two Hypothetical Protein Isoforms: RGA5-A and RGA5-B. (A) Structure of RGA5-A and RGA5-B transcripts. RGA5-A, as described by Okuyama et al. (2011), is produced by splicing of three introns from the primary RGA5 transcript. RGA5-B is produced by splicing of the first two introns and retention of intron 3. E1, exon 1; E2, exon 2; E3, exon 3; E4, exon 4. (B) RGA5-A and RGA5-B differ only in their C terminus. Intron retention in RGA5-B leads to a divergent C terminus (underlined) and disruption of the RATX1 domain present only in RGA5-A (marked in gray). In the rest of the proteins, including CC, NB, and LRR domains, as well as the first half of C-terminal non-LRR sequences, RGA5-A and RGA5-B are identical.

Figure 4.

RGA5-A Is the Functional Isoform Required for Pi-CO39 and Pia Resistance. A transgenic line of rice cultivar Kanto51 carrying RGA4 was transformed with a genomic construct for wild-type RGA5 (RGA5) or engineered genomic constructs for RGA5-A or RGA5-B (details in Supplemental Figure 2 online). Plants of the transgenic lines were spray inoculated with the transgenic strains Guy11-AVR1-CO39 or Guy11-EV or the field isolate INA72 carrying AVR-Pia (Okuyama et al., 2011), and symptoms were recorded until 7 d after inoculation. Only RGA4 RGA5-A and RGA4 RGA5 rice lines were resistant to M. oryzae strains expressing AVR1-CO39 or AVR-Pia. Presence of the transgenes was determined by direct PCR with RGA4- and RGA5-specific primers (bottom). Rice transformation was performed twice, and identical results were obtained in two independent inoculation experiments using at least 15 independent transgenic lines for each construct (see Supplemental Figure 3 online for the replicate experiment). Pictures show typical symptoms at 7 d after inoculation.

Figure 5.

AVR1-CO39 and AVR-Pia Interact with the C-Terminal Non-LRR Domain of RGA5-A in Yeast Two-Hybrid Assays. (A) RGA5-A_L and RGA5-B-L constructs carry the C-terminal non-LRR domain of RGA5-A or RGA5-B, respectively, in fusion with the Gal4 activation domain (AD:RGA5-A_883-1116 and AD:RGA5-B_883-1069). RGA5_ΔC and RGA5-A_S are C- and N-terminal deletions, respectively, of RGA5-A_L (AD:RGA5_883-1022 and AD:RGA5-A_982-1116). (B) Interaction of AVR1-CO39 and AVR-Pia (BD:AVR1-CO39 and BD:AVR-Pia) with RGA5 constructs was assayed by a yeast two-hybrid experiment. Empty-AD and empty-BD vectors were used as controls. Cultures of diploid yeast clones were adjusted to an OD of 0.2, and three dilutions (1/10, 1/100, and 1/1000) were spotted on synthetic TDO medium (-Trp/-Leu/-His supplemented with 3-amino-1,2,4-triazole) to assay for interactions and on synthetic double drop out (DDO) medium (-Trp/-Leu) to monitor proper growth. Photos were taken after 4 d of growth.

Figure 6.

AVR1-CO39 and AVR-Pia Interact with the C-Terminal Non-LRR Domain of RGA5-A in Planta. (A) RGA4:HA, RGA5:HA, AVR-Pia:CFP, AVR1-CO39:CFP, and GFP were transiently expressed in N. benthamiana leaves by Agrobacterium infiltration. Protein extracts were analyzed by immunoblotting with anti-GFP (α-GFP) and anti-HA antibodies (α-HA) (Input). In addition, immunoprecipitation was performed with anti-GFP beads (IP GFP) and analyzed by immunoblotting with anti-GFP antibodies for the detection of immunoprecipitated Avr proteins and with anti-HA antibodies for the detection of coprecipitated RGA4-A or RGA5. (B) 3HA:RGA5-A_L (RGA5-A_883-1116), AVR-Pia:CFP, AVR1-CO39:CFP, and PWL2:CFP were transiently expressed in N. benthamiana, and samples were analyzed as described in (A).

Figure 7.

AVR-Pia-H3 Does Not Interact with RGA5-A. (A) The H3 allele of AVR-Pia harbors two nonsynonymous polymorphisms generating the amino acid changes F24S and T46N. (B) Interaction of RGA5-A_L (AD:RGA5-A_883-1116) with AVR-Pia (BD:AVR-Pia), AVR-Pia-H3 (F24S, T46N) (BD:AVR-Pia-H3), and AVR-Pia-H3.1 (F24S) (BD:AVR-Pia-H3.1) was assayed by a yeast two-hybrid experiment. Empty-AD and empty-BD vectors were used as controls. All interactions were assayed on TDO medium (-Trp/-Leu/-His) in three independent experiments, which gave identical results.

Figure 8.

RGA5 and the Unrelated Pik-1 Resistance Protein Possess Homologous RATX1 Domains Involved in Specific Avr Protein Binding. (A) Alignment of RGA5-A and Pik-1 RATX1 domain sequences. (B) Interaction of AVR-Pia, AVR-Pik, and AVR1-CO39 (BD:AVR-Pia, BD:AVR-Pik, BD:AVR1-CO39, AD:AVR-Pia, and AD:AVR-Pik) with RGA5 C-terminal non-LRR sequences (BD:RGA5-A_L and AD:RGA5-A_L) or Pik-1 N-terminal sequences (BD:Pik-1_Nter and AD:Pik-1_Nter) was assayed by a yeast two-hybrid experiment. Empty-AD and empty-BD vectors were used as controls. Cultures of diploid yeast clones were adjusted to an OD of 2, and four dilutions (1/10, 1/100, 1/1000, and 1/10,000) were spotted on synthetic TDO medium (-Trp/-Leu/-His supplemented with 3-amino-1,2,4-triazole) to assay for interactions and on synthetic double dropout (DDO) medium (-Trp/-Leu) to monitor proper growth. Photos were taken after 4 d of growth.

Figure 9.

Phylogenetic Analysis Indicates That Independent Events Lead to the Integration of the RATX1 Domain into RGA5-A and Pik-1. Phylogenetic relationship of the RATX1 domains of RGA5-A, Pik-1, Pikp-1, pi5-3, and closely related homologous RATX1 proteins from rice, reconstructed using the neighbor-joining distance method based on the alignment shown in Supplemental Figure 9 and Supplemental Data Set 1 online. Node supports are given in percentage of 1000 bootstrap replicates. The topology shows the condense consensus tree of the 1000 bootstrap replicates, with nodes with a bootstrap support <50% being collapsed. Branch lengths are proportional to phylogenetic distances estimated from the JTT + G amino acid substitution model.