Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat

Abstract

Powdery mildew resistance gene Pm21, located on the chromosome 6V short arm of Haynaldia villosa and transferred to wheat as a 6VS·6AL translocation (T6VS·6AL), confers durable and broad-spectrum resistance to wheat powdery mildew. Pm21 has become a key gene resource for powdery mildew resistance breeding all over the world. In China, 12 wheat varieties containing Pm21 have been planted on more than 3.4 million hectares since 2002. Pm21 has been intractable to molecular genetic mapping because the 6VS does not pair and recombine with the 6AS. Moreover, all known accessions of H. villosa are immune to powdery mildew fungus. Pm21 is still defined by cytogenetics as a locus. In the present study, a putative serine and threonine protein kinase gene Stpk-V was cloned and characterized with an integrative strategy of molecular and cytogenetic techniques. Stpk-V is located on the Pm21 locus. The results of a single cell transient expression assay showed that Stpk-V could decrease the haustorium index dramatically. After the Stpk-V was transformed into a susceptible wheat variety Yangmai158, the characterized transgenic plants showed high and broad-spectrum powdery mildew resistance similar to T6VS·6AL. Silencing of the Stpk-V by virus-induced gene silencing in both T6VS·6AL and H. villosa resulted in their increased susceptibility. Stpk-V could be induced by Bgt and exogenous H(2)O(2), but it also mediated the increase of endogenous H(2)O(2), leading to cell death and plant resistance when the plant was attacked by Bgt.

Fig. 1.

Mapping of Stpk-V by FISH and sequential GISH on mitotic metaphase chromosomes of T6VS·6AL and del6VS-1. (A and C) FISH with 30-kb TAC clone as the probe, and the green signal shows the Stpk-V position. (B and D) Sequential GISH with the genomic DNA of H. villosa as the probe. The arrows in A and B show the deletion 6V chromosomes, and in C and D they indicate the translocation chromosomes.

Fig. 2.

Functional analysis of Stpk-V by stable transformation into the susceptible wheat variety Yangmai158. (A) Detached leaves of the T₀ plants showed HRs 7 d after inoculation with Bgt, whereas the leaves of Yangmai158 were covered with colonies. (B) T₁ plant without Stpk-V (T_1(S)) showed susceptibility, whereas the T₁ plant with Stpk-V (T_1(R)) showed high resistance in the greenhouse. (C) T₃ lines showed a similar high level of resistance in the field as T6VS·6AL. T₃-6, resistant line with hypersensitive mosaics, T₃-2, resistant line without hypersensitive mosaics.

Fig. 3.

Functional analysis of Stpk-V by VIGS. (A) Quantitative RT-PCR analysis of the expression level of Stpk-V in the plants inoculated with BSMV:Stpk-V using the plants inoculated with BSMV:GFP as the control (CK). In seven BSMV:Stpk-V–inoculated H. villosa plants (A1) and seven BSMV:Stpk-V–inoculated T6VS·6AL plants (A2), the expression level of Stpk-V was decreased. (B) Observations of the development of Bgt in the BSMV:Stpk-V–inoculated plants using the BSMV:GFP inoculated plants as the control. H. villosa (B1) and T6VS·6AL (B3) were infected with BSMV:GFP and then inoculated with Bgt. Infection with BSMV:GFP did not alter the resistance to Bgt. H. villosa (B2) and T6VS·6AL (B4) infected with BSMV:Stpk-V and then inoculated with Bgt. Stpk-V silencing resulted in increased susceptibility. Arrows indicate spore (Sp), appressorium (App), secondary hyphae (SH), and conidiophore (CH).

Fig. 4.

Characterization of the resistance mechanism of Stpk-V by observing of H₂O₂ and HRs after Bgt inoculation in leaves of Yangmai158 (S), T6VS·6AL (R), and the transgenic plants (T). (A) In Yangmai158, Bgt developed normally. H₂O₂ was localized around the primary penetration peg at 6 h (S-6) and then appeared under the newly developed penetration pegs at 48 h (S-48). At 120 h, the leaves were covered with hyphae and conidiophores (S-120). (B) In the T6VS·6AL, there were two types of Bgt development patterns. For the first type, the development of Bgt stopped after App formation. H₂O₂ was detected in the interaction cell (R-6), and the HR was then observed at 24 h (R-24). For the second one, the haustoria could form (R′-24), and the hyphae elongated, but very slowly (R′-120). (C) In the transgenic plants, Bgt could not develop after App formation. H₂O₂ was detected in the interaction cell (T-6), and the HR was observed at 24 h (T-24). Arrows indicate spore (Sp), appressorium (App), haustoria (Ha), secondary hyphae (SH), HR cell (HR), and conidiophore (CH). Numbers indicate the hours of Bgt inoculation. (Scale bar, 25 μm.)