Identification and transfer of a new Pm21 haplotype with high genetic diversity and a special molecular resistance mechanism

Abstract

A new functional Pm21 haplotype, Pm21(8#), was cloned from the new wheat-H. villosa translocation line T6VS(8#)·6DL, which confers the same strong resistance to powdery mildew through a different resistance mechanism. Broad-spectrum disease resistance genes are desirable in crop breeding for conferring stable, durable resistance in field production. Pm21(4#) is a gene introduced from wild Haynaldia villosa into wheat that confers broad-spectrum resistance to wheat powdery mildew and has been widely used in wheat production for approximately 30 years. The discovery and transfer of new functional haplotypes of Pm21 into wheat will expand its genetic diversity in production and avoid the breakdown of resistance conferred by a single gene on a large scale. Pm21(4#) previously found from T6VS(4#)·6AL has been cloned. In this study, a new wheat-H. villosa translocation, T6VS(8#)·6DL, was identified. A new functional Pm21 haplotype, designated Pm21(8#), was cloned and characterized. The genomic structures and the splicing patterns of Pm21(4#) and Pm21(8#) were different, and widespread sequence diversity was observed in the gene coding region and the promoter region. In the field, Pm21(8#) conferred resistance to Blumeria graminis f. sp. tritici (Bgt), similar to Pm21(4#), indicating that Pm21(8#) was also a resistance gene. However, Bgt development during the infection stage was obviously different between Pm21(4#)- and Pm21(8#)-containing materials under the microscopic observation. Pm21(4#) inhibited the formation of haustoria and the development of hyphae in the initial infection stage, while Pm21(8#) limited the growth of hyphae and inhibited the formation of conidiophores in the late infection stage. Therefore, Pm21(8#) is a new functional Pm21 haplotype that provides a new gene resource for wheat breeding.

Fig. 1

Cytogenetic identification and molecular marker analysis of the new wheat-H. villosa translocation T6VS(8#)·6DL, a–b Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) analysis of T6VS(5#)·6DL and T6VS(8#)·6DL on root tip metaphase chromosomes. Green fluorescence represents GISH analysis signal obtained using the total genomic DNA of H. villosa as a probe, and the red fluorescence represents the FISH analysis signal obtained using oligo-pAs as a probe. Chromosomes were counterstained with DAPI (blue). The translocation chromosomes are indicated with white arrows. c–d GISH-FISH patterns of the D-genome chromosomes of the two translocation lines. The chromosomes were extracted from a and b respectively, and the two translocation chromosomes, 6VS(5#)·6DL and 6VS(8#)·6DL, were identified and are indicated. e PCR analysis of the codominant molecular marker CINAU15. The chromosome specific bands are labeled on the right, and the band of 6VS(8#) was different from that of 6VS(4#) or 6VS(5#) (color figure online)

Fig. 2

Evaluation of the powdery mildew resistance in the wheat-H. villosa translocation line T6VS(8#)·6DL, a Powdery mildew resistance evaluation in T6VS(8#)·6DL at the seedling stage in the greenhouse, using T6VS(4#)·6AL and T6VS(5#)·6DL as the resistant controls and Nannong0686 as the susceptible control. b Powdery mildew resistance evaluation in T6VS(8#)·6DL at the adult plant stage in the field, using T6VS(5#)·6DL as the resistant control

Fig. 3

Functional analysis of Pm21(8#) using BSMV-VIGS, a Evaluation of Bgt resistance conferred by Pm21(8#) in T6VS(8#)·6DL by silencing of Pm21(8#). Seven days after Bgt inoculation, no Bgt colonies were visible on the BSMV:γ infected leaves and on the BSMV:PDS infected leaves, while a small number of Bgt colonies were visible on the BSMV:Pm21(8#)-infected leaves, and a large number of Bgt colonies were visible in the susceptible control Nannong0686. b qRT‒PCR analysis of Pm21(8#) expression levels in both BSMV:γ and BSMV: Pm21(8#)-infected individuals. The expression level of Pm21(8#) was decreased significantly in the BSMV:Pm21(8#) infected leaves. c–h Observation of Bgt development under the microscope. Bgt developed more slowly and produced fewer hyphal branches in the BSMV:γ and BSMV:PDS infected leaves than in the BSMV:Pm21(8#)-infected leaves (c and d). Moreover, a large number of conidial chains could be produced in the BSMV:Pm21(8#)-infected leaves but not in the BSMV:γ and BSMV:PDS-infected leaves (e and g). Figures f and h show enlarged views of the red rectangles in e and g, respectively. The white line represents the scale bar 200 μm

Fig. 4

Comparison of the gene structures and protein compositions of the Pm21(4#), Pm21(5#) and Pm21(8#), a Gene structures of Pm21(4#), Pm21(5#) and Pm21(8#) by alignment of the genomic sequences with the corresponding transcripts. An alternative splicing pattern was identified in Pm21(8#) but not in Pm21(4#) and Pm21(5#). Exons are represented by black rectangles, and introns are represented by lines. Their sizes are marked in bp. b Protein compositions of Pm21(4#), Pm21(5#), and Pm21(8#) based on searching the conserved motifs. Pm21(4#), Pm21(5#) and Pm21(8#)-1 were complete NLR proteins, while Pm21(8#)-2 was a truncated protein with a premature stop codon at 200 aa. The Rx_N (CC), NB-ARC and LRR domains were identified respectively, and each conserved motif distributed in the three domains is displayed in a different color (color figure online)

Fig. 5

Observation of the Bgt development on the leaves of the three translocation lines at different time points after Bgt inoculation, An obvious difference was detected in T6VS(8#)·6DL, in which more conidia could produce hyphae. On the left side of the figure, 24 hpi, 48 hpi, 72 hpi and 120 hpi are the hours post Bgt inoculation, respectively. The white line represents the scale bar 100 μm

Fig. 6

Detection of ROS accumulation by DAB staining of Bgt-infected leaves, An obvious difference was detected in materials containing Pm21(8#), including T6VS(8#)·6DL and F₂-Pm21(8#), in which fewer Bgt-interacting cells showed ROS accumulation. In the up portion of the figure, 24 hpi and 48 hpi are the hours post Bgt inoculation. The white line represents the scale bar 2 mm