No abstract

Pattern recognition receptors (PRRs) can trigger plant immunity through the recognition of pathogen-associated molecular patterns. In this study, we report that a malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, from Haynaldia villosa putatively acts as a PRR to positively regulate powdery mildew resistance caused by Blumeria graminis f. sp. tritici (Bgt) in wheat. RLK-V has two alternatively spliced transcripts corresponding to an intact RLK-V1.1 and a truncated RLK-V1.2 caused by intron retention. Expression analysis showed that both transcripts could be up-regulated by Bgt in resistant materials, whereas the functional RLK-V1.1 was expressed only after Bgt inoculation. Promoter activity assays indicated that RLK-V could respond to Bgt even in susceptible wheat. Silencing of RLK-V in Pm21-carrying resistant materials resulted in compromised resistance to Bgt. In addition, over-expression of RLK-V1.1 in Pm21-lacking susceptible Yangmai158 and SM-1 by single-cell transient expression and stable transformation in Yangmai158 could improve powdery mildew resistance. We propose that RLK-V regulates basal resistance to powdery mildew, which is also required for broad-spectrum resistance mediated by the Pm21 gene. Over-expression of RLK-V1.1 could trigger cell death in Nicotiana benthamiana, and RLK-V1.1 transgenic wheat accumulated more reactive oxygen species and displayed a stronger hypersensitive response than did the recipient, which led to enhanced Bgt resistance. However, constitutive activation of RLK-V1.1 resulted in the abnormal growth of transgenic plants.

Wheat powdery mildew caused by Blumeria graminisb f. sp. tritici is one of the most destructive diseases all over the world. Pm21, transferred from the wild Haynaldia villosa to wheat, confers broad spectrum resistance throughout the whole stage, and this gene has been widely used in wheat production for more than 20 years. Cloning the candidate gene of Pm21 is the prerequisite for elucidating the resistance mechanism, and is a valuable attempt to clone the target genes from the evolutionarily distant wild species. In this study, an innovative approach, which combined cytogenetic stocks development, mutagenesis, RenSeq and PacBio, was tried successfully to clone an NBS-LRR type gene NLR1-V from the Pm21 locus. Firstly, a powdery mildew resistant cryptic alien introgression line HP33 involved very small 6VS segment was developed, and 6 independent susceptible mutants of T6VS· 6AL was identified. Then, the transcriptome of H. villosa was obtained by NGS and the full-length NBS-LRR gene database was constructed by RenSeq-PacBio. In the following study, two expressed NLR genes were located to the Pm21 locus using the HP33 as the mapping material, and only NLR1-V showed polymorphism between the wild T6VS· 6AL and its six mutants. The functional analysis indicated that silencing of NLR1-V could compromise the resistance of T6VS· 6AL completely, and could also decrease the resistance of T6VS· 6DL dramatically. Moreover, NLR1-V could recover the resistance of the susceptible mutant and increase the resistance in the susceptible wheat. The study implied that NLR1-V, a CC-NBS-LRR encoding gene, is a potential candidate gene of the powdery mildew resistance gene Pm21.