Pb1

The rice Panicle blast 1(Pb1) gene is well known as the panicle blast resistance gene.

Annotated Information

" Figure 1a. Pb1 is a panicle blast-resistance gene of broad spectrum for fungal races. Disease symptoms of cultivar Koshihikari-Aichi-SBL (Pb1+) and near isogenic Koshihikari (Pb1-), grown in an experimental paddy field at the Aichi Agricultural Research Center field (left) and close-ups of their panicles (right).The panicle resistance was evaluated quantitatively by percentage diseased grains (lower panel)(from reference )"

" Figure 1b. Pb1 is a panicle blast-resistance gene of broad spectrum for fungal races. Race specificity of Pb1-based blast resistance. Aichinokaori-SBL (Pb1+) and near-isogenic Aichinokaori (Pb1-) were inoculated with 16 Japanese blast fungus isolates (triangle) including 14 Japanese race standard isolates(Hayashi, 2005) or seven isolates of foreign origin (•). The blast isolates are numbered in black (Japanese) or red (foreign) letters.(from reference )"

" Figure 2. Developmental pattern ofPb1expression and blast resistance. The developmental pattern ofPb1expression relative toOsUbi1in the leaves of Pb1+ cultivar Koshihikari-Aichi-SBL at the 2 (2L), 6 (6L), 10 (10L) and flag leaf(FL) stages, and in the panicle after heading, are shown by dotted lines.Pb1 mRNA levels shown are relative to those of riceubiquitin 1(Os06g0681400). Spores of blast fungus isolate 93–406 (race 001.0), a race compatible with Nipponbare, were spray-inoculated at a concentration of 0.2–2.5×105 spores ml-1. Disease symptoms were evaluated by lesion numbers at the 2, 6, 10 and flag leaf stages, and the percentage of diseased grains relative to those in near-isogenic Koshihikari without Pb1. (from reference )"

" Figure 3.Pb1 specifically interacts with WRKY45. (A) Y2H assay. Interaction between Pb1 and several rice WRKY proteins was examined with the Nterminal region of Pb1 (Pb1-CC1–43) as bait and the N-terminal regions of WRKY TFs as prey. (B) GST pull-down assay. Wild-type and mutant Pb1-CCs were tested for interaction with full-length HA-WRKY45. Pb1-CCs of wild type (Pb1-CC1–43 and Pb1-CC1–51) and mutants that had two (Pb1-CC1– 5116A23A and Pb1-CC1–5130A37A) or four (Pb1-CC1–51Quad) amino acid substitutions were tested. Pb1-CC1–5130A37A shows somewhat stronger interaction, but this result was not reproducible. Two bands detected for HAWRKY45 are due to different phosphorylation states (20). Zinc staining of the gel is shown in Lower.(C) Coimmunoprecipitation assay. Full-length Pb1-HASt and myc-WRKY45 were expressed in a wheat germ extract and analyzed by coimmunoprecipitation. (D) Split luciferase assay. NRluc-Pb1 or NRluc-Pb1-Quad were coexpressed with C-RLuc-WRKY45 in rice protoplasts and assayed for reconstituted luciferase activity. Mean activities for three independent samples are shown with SD )"

"Pb1 figure 4. Pb1-mediated panicle blast resistance is WRKY45 dependent. (A)Effects of WRKY45 knockdown on panicle blast resistance. The plants were spray inoculated with blast fungus conidia (1–3×105mL−1) at full-heading stage and evaluated for disease symptoms by percent diseased grains.Averages of 20 plants each are shown with SEs. The experiments were performed four times with similar results. (B) Effects of WRKY45 knockdown on leaf-blast resistance byPb1overexpression. The plants were spray inoculated with blast fungal conidia (6×104mL−1) and evaluated for diseased leaf area at 11 dpi. Average lesion areas (%) in 20 plants are shown with SE.The experiments were performed four times with similar results. NB, Nipponbare (C)Pb1–WRKY45 interaction is required for blast resistance. The experiments were performed three times with similar results. (D) Pb1-dependent blast resistance is mainly due to preinvasive defense. The plants were inoculated with rice blast fungus by leaf sheath inoculation and examined for the rate of invasion into rice at 2 dpi. Means are shown with SD. )"

"Pb1 figure 5.Pb1 protects WRKY45 proteins from degradation. (A)Coexpression of Pb1 enhanced WRKY45 accumulation in wheat germ extract. HA-WRKY45 was translated in a wheat germ extract in the absence (control) or presence of MG132, or cotranslated with Pb1, and detected by Western blot. The experiments were performed three times with similar results. (B)Coexpression of Pb1-Quad induced less WRKY45 accumulation. Myc-WRKY45 was cotranslated with Pb1-HASt or Pb1-Quad-HASt in wheat germ extracts by using transcripts of myc-WRKY45(0.5μL) and increasing amounts of Pb1-HASt or Pb1-Quad HASt transcript. The proteins were detected by Western blot. (C) Pb1 enhances WRKY45 accumulation in rice protoplasts. Rice protoplasts were transfected with PUbi:myc-WRKY45(1μg) together with increasing amounts of HA-tagged Pb1, Pb1-Quad, or Pb1-NES plasmids. The proteins were detected by Western blot. (D) Transactivation assay. The reporter construct contained four W-boxes (15, 37) upstream of the NanoLuc coding sequence. The reporter,effector, and reference (35S:hRLUC) plasmids were delivered into the leaf sheath by particle bombardment. NanoLUC activities were determined and normalized to the reference hRLUC activity. Mean activities of four independent samples are shown in arbitrary units with SD. Student’sttests indicated that the activities with Pb1-Quad and Pb1-NES were significantly lower than that with Pb1 (P<0.05) and that there is no significant change in the activity compared with that with WRKY45 only.)"

Function

 Panicle blast 1(Pb1) is a panicle blast resistance gene derived from the indica rice cultivar “Modan”. Pb1 encodes a coiled-coil–nucleotide-binding site–leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Its molecular mechanism is  that CC-NB-LRR protein Pb1 interacts with WRKY45, a key transcription factor of the SA pathway, and that the blast resistance by Pb1 depends on WRKY45 .

Pb1 is a broad-spectrum panicle blast-resistance gene and Expression pattern of Pb1 accounts for adult resistance in Pb1 lines

 Even if rice plants survive leaf blast, they can be damaged by panicle blast in the field (Figure 1a,Pb1-). Rice cultivars with Pb1(Pb1+  cultivars) are rather weakly resistant to leaf blast, however, they show remarkable resistance to panicle blast (Figure 1a, Pb1+). Evaluation of diseased grains indicated that the resistance was of quantitative nature (Figure 1a).
 To examine the race specificity of Pb1-based panicle blast resistance, a Pb1+cultivar, Aichinokaori-SBL (Pb1+), and its near-isogenic cultivar, Aichinokaori (Pb1-), were inoculated with various isolates of blast fungus at the full heading stage. Evaluation of diseased grains 2 weeks after inoculation showed that Aichinokaori-SBL (Pb1+) was more resistant to panicle blast than Aichinokaori (Pb1-) with all the blast isolates tested (Figure 1b). This demonstrates the broad-spectrum nature of Pb1-based panicle blast resistance
 In a Pb1+ cultivar Koshihikari-Aichi-SBL, the level of blast resistance increased with plant growth. The resistance was weak during young stages, but became stronger in adult stages (adult resistance and panicle resistance, Figure 2). the expression pattern of Pb1 is a determinant for the developmental pattern of blast resistance in Pb1+ cultivars

Pb1 Interacts with WRKY45 Through Its CC Domain, Prevents Ubiquitin-Proteasome Degradation of WRKY45 and its Blast Resistance Depends on WRKY45

 Pb1-CC interacted with only WRKY45, but not with other WRKY proteins including WRKY47, another group III WRKY protein, in yeast cells (Figure. 3A). The interaction was also observed between wild-type Pb1-CC (Pb1-CC1–44and Pb1-CC1–51) and full ength WRKY45 in a GST-pull down assay (Figure. 3B). Mutants of Pb1-CC that had mutations in the two hydrophobic amino acids (Pb1-CC1–5116A23A and Pb1-CC1–5130A37A) interacted with WRKY45 similarly to wild-type Pb1-CC1–51,Pb1-16A23A30A37A (Quad), in which the four hydrophobic amino acids were mutated, showed markedly decreased interaction with WRKY45 in comparison with its corresponding wild-type (Pb1-CC1–51) (Figure. 3B). A coimmunoprecipitation assay showed that full-length Pb1 interacted with full-length WRKY45, but Pb1-Quad (full-length) interacted very weakly (Figure. 3C). This Pb1–WRKY45 interaction and the reduction of the interaction by CC mutation were also observed in a split LUC system by using N-terminal and C-terminal luciferases fused with Pb1 and WRKY45, respectively, in rice leaf sheath cells (Figure. 3D)
 To examine whether the panicle blast resistance by Pb1 depends on WRKY45, WRKY45 knockdown (WRKY45-kd) lines (Pb1+/WRKY45-kd and Pb1–/WRKY45-kd) were constructed. WRKY45 knockdown did not affect Pb1expression in these transformants. But the levels of panicle resistance in Pb1–/WRKY45-kd plants were similar to those in the Pb1–(Koshihikari) line, indicating that WRKY45 knockdown did not affect the basal levels of blast resistance in the panicle (Pb1 figure 4A)
 Generating rice transformants that overexpressed Pb1 in a WRKY45-kd Nipponbare background (Pb1-ox/WRKY45-kd). Leaf blast resistance tests showed that the resistance by Pb1 overexpression was largely compromised by WRKY45 knockdown (Pb1 figure 4B). In rice transformants overexpressing Pb1-Quad in Nipponbare (Pb1-Quad-ox), leaf blast resistance was weaker than in Pb1-ox plants expressing wild-type Pb1at comparable levels (Pb1 figure 4C). Collectively, these results indicated that WRKY45 is required for Pb1-mediated blast resistance both in the panicle and the leaf and suggested that direct interaction with WRKY45 through the CC domain is required for the resistance
 Fungal invasion into rice cells was observed from only 15–20% of fungal conidia, whereas the invasion rate was 80% in Nipponbare. The invasion rate into Pb1-ox/WRKY45-kd rice was comparable to Nipponbare (Pb1 figure 4D). These results were also consistent with WRKY45 dependence of the blast resistance by Pb1.
 Coexpression of Pb1 stabilized WRKY45 proteins in germ extract (Pb1 figure 5A). Coexpression of Pb1-Quad resulted in smaller amounts of WRKY45 proteins (Pb1 figure 5B)
 To investigate this phenomenon,myc-WRKY45was transiently expressed in rice protoplasts and the effects of Pb1 coexpression on the WRKY45 protein levels were tested. As shown in Pb1 figure 5C, myc-WRKY45 was accumulated by coexpression of Pb1 and, to a lesser extent, by coexpression of Pb1-Quad or Pb1-NES. These results suggested that Pb1 protected WRKY45 proteins from UPS dependent degradation through a protein–protein interaction.
 The effects of Pb1 coexpression on transcriptional activities by WRKY45 were examined in a transient transactivation assay by using rice sheath cells. Coexpression of Pb1 enhanced the luciferase activity due to WRKY45 by approximately fourfold (Pb1 figure 5D). In contrast, coexpression of Pb1-Quad or Pb1-NES enhanced the activity by only twofold (Pb1 figure 5D).

Mutation

Pb1-Quad is a coiled-coil domain mutant that had weak interaction with WRKY45. Pb1+/WRKY45-kd is a WRKY45 knock down line Pb1–/WRKY45-kd is a line that Pb1 and WRKY45 were both knock down

Expression

Pb1 figure 6.Pb1 protein sequence and conserved motifs. Protein sequence of Pb1. Relatively well-conserved sequence motifs with rice R proteins are shown in blue letters and less conserved ones in green letters. An LRR domain is underlined. The position of an extra E in P5 is indicated by a red triangle. Genomic DNA and protein sequences were deposited in DDBJ with accession numbers of AB570371 (Pb1) and AB570370 (from reference ).

Pb1 encodes a coiled-coil–nucleotidebinding-site–leucine-rich repeat (CC–NBS–LRR) protein. The Pb1 protein sequence differed from previously reported R-proteins, particularly in the NBS domain, in which the P-loop was apparently absent and some other motifs were degenerated. Pb1 has two putative CC domains, CC1 and CC2, located in its N-terminus, with an nT motif-like sequence intervening them.CC domains have periodical occurrence of leucine, or other hydrophobic amino acids, showing extensive amino acid sequence similarity to the CC domains of other CC-NBS-LRR proteins.An NBS-like domain is present in the central region of Pb1 protein; however, its sequence is quite diverged from those in reported NBS-LRR proteins.A long stretch of polypeptide with no significant sequence similarity to other sequences is inserted after the CC region of Pb1 and A Walker B-like sequence is present at amino acid position 641 of Pb1

Knowledge Extension

Pb1-mediated blast resistance partially depends on the SA signaling pathway. Two possible mechanisms can account for the partial SA dependence of Pb1-mediated blast resistance. One is that Pb1 activates the SA pathway upstream of SA as many R proteins do , leading to WRKY45 induction through the SA

pathway. Another is that reduction of basal SA levels results in a decrease of basal WRKY45 protein levels, which consequently leads to reduced accumulation of WRKY45 proteins despite the inhibition of WRKY45 degradation by Pb1.

Labs working on this gene

Aichi prefectural Agriculture Research Center(AARC),1-1 Sagamine, Yazako, Nagakute, Aichi 480-1193, Japan(Fujii-K,Sugiura-N,Tsuji-T,Izawa-T) Hokkaido National Agriculture Experiment Station,1 Hitsujigaoka, Toyohira-ku, Sapporo, Hokkaido 062-8555, Japan( Hayano-Saito-Y, Saito-K, Iwasaki-M) Mountainous Regional Agriculture Research Institute, AARC,11 Susogaeto, Inabu, Aichi 441-2513, Japan(Hayashi-N) Plant Disease Resistance Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan(Nagao Hayashi, Haruhiko Inoue, Takashi Matsumoto, Masahiro Yano, Hiroshi Takatsuji) Field Crop Division, Aichi Agricultural Research Center, Nagakutecho, Aichi 480-1103, Japan( Takahiro Kato, Taketo Funao, Masaki Shirota,) Institute of the Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Ippaizuka, Tsukuba,Ibaraki 305-0854, Japan(Takehiko Shimizu, Hiroyuki Kanamori, Hiroko Yamane) Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.

References

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