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 )"

[[File::figure 1b.jpg|left|thumb|200px|" 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 (4) 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  )"]]

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

 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

Expression pattern of Pb1 accounts for adult resistance in Pb1 lines

 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 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 (Fig. 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 (Fig. 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) (Fig. 3B). A coimmunoprecipitation assay showed that full-length Pb1 interacted with full-length WRKY45, but Pb1-Quad (full-length) interacted very weakly (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 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 (Fig.4D). These results were also consistent with WRKY45 dependence of the blast resistance by Pb1.

Pb1 Prevents Ubiquitin-Proteasome Degradation of WRKY45

 Coexpression of Pb1 stabilized WRKY45 proteins in germ extract (Fig. 5A). Coexpression of Pb1-Quad resulted in smaller amounts of WRKY45 proteins (Fig. 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 Fig. 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 (Fig. 5D). In contrast, coexpression of Pb1-Quad or Pb1-NES enhanced the activity by only twofold (Fig. 5D).

GO assignment(s): GO:0005506, GO:0016216, GO:0017000

References

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