Cysteine-rich secretory proteins (C), antigen 5 (A), and pathogenesis-related 1 proteins (P) comprise widespread CAP superfamily proteins, which have been proven to be novel virulence factors of mammalian pathogenic fungi and some plant pathogens. Despite this, the identification and function of CAP proteins in more species of plant pathogens still need to be studied. This work presents the identification and functional analysis of CAP superfamily proteins from Puccinia striiformis f. sp. tritici (Pst), an important fungal pathogen that causes wheat stripe rust on wheat worldwide. A total of six CAP genes were identified in the Pst genome, designated as PsCAP1–PsCAP6. Five PsCAP proteins, including PsCAP1, PsCAP2, PsCAP3, PsCAP4, and PsCAP5, have N-terminal signal peptides secreted with the yeast signal sequence trap assay. Single-nucleotide polymorphism (SNP) analysis indicated that they showed a low level of intraspecies polymorphism. The expression abundance of PsCAP genes at different Pst infection stages was detected by RT-qPCR, and most of them were highly expressed during Pst infection on wheat and also Pst sexual reproduction on barberry (Berberis shensiana). Noticeably, the silencing of these six PsCAP genes by BSMV-mediated HIGS indicated that PsCAP1, PsCAP4, and PsCAP5 contribute significantly to Pst infection in wheat. These results indicate that PsCAP proteins may act as virulence factors during Pst infection, which also provides insights into Pst pathogenicity.

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus from P. striiformis strain GS-1, which was designated as “Puccinia striiformis mitovirus 2” (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 multiplied highly during Pst infection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 in Pst by barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity of Pst. These results indicate PsMV2 promotes host pathogenicity in Pst. Interestingly, PsMV2 was detected among a wide range of field isolates of Pst and may have coevolved with Pst in earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution in Pst which may offer new strategies for disease control.

Wheat leaf rust, caused by the biotrophic fungus Puccinia triticina ( Pt ), is a major threat to global wheat production. Fungal pathogens often deploy plant cell wall-degrading enzymes to breach host barriers, with glycoside hydrolases (GHs) providing hydrolytic activity and carbohydrate-binding modules (CBMs) enabling substrate recognition. However, research on their specific roles in the leaf rust fungus remains limited. Here, we functionally characterized two Pt genes, PtGH1 and PtCBM1 . PtCBM1, a carbohydrate-binding module protein, binds cellulose and potentiates cellulase activity despite lacking hydrolase activity, whereas PtGH1 encodes a β-glucanase secreted via a non-classical pathway. Both proteins suppress Bax-induced cell death in Nicotiana benthamiana , suggesting immune-suppressive activity. Silencing either of the two genes in wheat via host-induced gene silencing significantly reduced fungal virulence, impaired hyphal growth, and enhanced host defense. Together, these findings identify PtGH1 and PtCBM1 as distinct virulence factors that act through complementary mechanisms, involving physical facilitation and enzymatic degradation of host cell walls. This dual strategy illustrates how the leaf rust fungus overcomes host immunity and provides potential molecular targets for developing durable wheat resistance.