Puccinia striiformis f. sp. tritici (Pst) is an important obligate pathogen in wheat (Triticum aestivum L.) and secretes effectors into plant cells to promote infection. Identifying host targets of effector proteins and clarifying their roles in pathogen infection is essential for understanding pathogen virulence. In this study, we identified a serine-rich effector, Pst27791, from Pst that suppresses cell death in Nicotiana benthamiana. Stable overexpression of Pst27791 in wheat suppressed reactive oxygen species accumulation and the salicylic acid-dependent defense response. Transgenic wheat expressing the RNA interference construct of Pst27791 exhibited high resistance to Pst virulent isolate CYR31, indicating its importance in pathogenesis. Pst27791 interacting with wheat rapidly accelerated fibrosarcoma (Raf)-like kinase TaRaf46 in yeast and in planta. Knocking down TaRaf46 expression in wheat attenuated Pst infection and increased wheat immunity. The overexpression of TaRaf46 decreased wheat resistance to Pst and repressed MAPK activation in wheat. Pst27791 may stabilize TaRaf46 through the inhibition of proteasome-mediated degradation in N. benthamiana. The ability of Pst27791 to enhance Pst colonization was compromised when TaRaf46 was silenced, suggesting that the virulence of Pst27791 is mediated by TaRaf46. Overall, these results indicate that Raf-like kinase TaRaf46 is exploited by the Pst effector as a negative regulator of plant immunity to promote infection in wheat.

Jujube witches’ broom (JWB) disease, caused by Candidatus Phytoplasma ziziphi (Ca. P. ziziphi), severely threatens the production of Chinese jujube (Ziziphus jujuba Mill.). Emerging evidence highlights the critical role of phytoplasma-secreted effectors in pathogenesis, though few have been functionally characterized. Here, we identified a Sec-dependent effector, JWB790, from Ca. P. ziziphi, which was shown to suppress plant immunity. Through transient expression assays in Nicotiana benthamiana, pathogen inoculation assays, the generation of transgenic Arabidopsis thaliana plants, and RNA-seq-based transcriptomic profiling, we systematically investigated the virulence function of JWB790. Our findings revealed that JWB790 is highly expressed in JWB-infected tissues. The transient expression of JWB790 in N. benthamiana suppressed BAX-induced cell death and H₂O₂ accumulation. Furthermore, the stable overexpression of JWB790 in A. thaliana compromised disease resistance, accompanied by reduced H₂O₂ accumulation and callose deposition triggered by flg22. Additionally, the RNA-seq analysis of JWB790 transgenic Arabidopsis plants indicated that the overexpression of JWB790 altered the expression of biotic stress-related genes. In summary, JWB790 is a virulence factor that suppresses plant immunity and promotes pathogen proliferation. These results advance our understanding of Ca. P. ziziphi pathogenesis.

The thymidylate kinase (tmk) gene is indispensable for the proliferation and survival of phytoplasma. To reveal the molecular variation and phylogeny of the tmk genes of Candidatus phytoplasma ziziphi, in this study, the tmk genes of 50 phytoplasma strains infecting different resistant and susceptible jujube cultivars from different regions in China were amplified and analyzed. Two sequence types, tmk-x and tmk-y, were identified using clone-based sequencing. The JWB phytoplasma strains were classified into three types, type-X, type-Y, and type-XY, based on the sequencing chromatograms of the tmk genes. The type-X and type-Y strains contained only tmk-x and tmk-y genes, respectively. The type-XY strain contained both tmk-x and tmk-y genes. The type-X, type-Y, and type-XY strains comprised 42%, 12%, and 46% of all the strains, respectively. The type-X and type-XY strains were identified in both susceptible and resistant jujube cultivars, while type-Y strain was only identified in susceptible cultivars. Phylogenetic analysis indicated that the tmk genes of the phytoplasmas were divided into two categories: phylo-S and phylo-M. The phylo-S tmk gene was single-copied in the genome, with an evolutionary pattern similar to the 16S rRNA gene; the phylo-M tmk gene was multi-copied, related to PMU-mediated within-genome transposition and between-genome transfer. Furthermore, the phylogenetic tree suggested that the tmk genes shuttled between the genomes of the Paulownia witches’ broom phytoplasma and JWB phytoplasma. These findings provide insights into the evolutionary and adaptive mechanisms of phytoplasmas.

Peony (Paeonia suffruticosa Andr.), belonging to family Paeoniaceae, is an important medicinal and ornamental plant. During August of each year from 2016 to 2023, peony plants at Heze city were found to exhibit leaf yellows symptoms. The incidence rate of the symptomatic plant was recorded from 10% to 30% in four peony gardens with about 200 acres. Total DNA was extracted from 0.10 g fresh plant leaf tissues from 24 symptomatic and 8 asymptomatic samples using rapid plant genomic DNA isolation kit (Aidlab Biotechnology, Beijing, China). The extracted DNA was amplified by nested polymerase chain reaction using universal primers R16mF2/R16mR1 followed by R16F2/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) specific for the 16S rRNA gene and new designed tuf gene specific primers JWB-tuforfF1 (5'-ATGGCTGAAATATTTTCAAGAG-3') and JWB-tuforfR1 (5'-TTATTCTATGATTTTAATAACAG-3') followed by JWB-tuforfF2 (5'-ATGTAAACGTAGGAACTATTGG-3') and JWB-tuforfR2 (5'- TCCGATAGTTCTTCCACCTTCAC-3'). Amplicons of about 1.25 kb and 1.02 kb (16S rRNA gene and tuf gene, respectively) were obtained in 8 symptomatic samples from four peony gardens. However, no amplification was obtained in any of the asymptomatic samples. The representative amplicons of 16S rRNA and tuf genes of three positive samples (Heze-9, -18 and -27) were cloned into a zero background pLB-simple vector (Tiangen Biotechnology, Beijing, China) and sequenced by Taihe Biotechnology, Beijing, China. Sequences obtained in the study were deposited in NCBI GenBank with accession numbers PP504882, PP504883 and PP504884 for the 16S rRNA gene as well as PP530237, PP530238 and PP530239 for the tuf gene. The phytoplasma strain under the study was described as peony yellows (PeY) phytoplasma, PeY-Heze strain. Alignment analysis by DNAMAN software showed that three 16S rRNA gene sequences obtained in the study shared 99.36% to 99.60% sequence identity and three tuf gene sequences obtained in the study were identical. BLAST analysis of the 16S rRNA gene sequences of the PeY-Heze phytoplasma strains showed 99.60%-99.84% sequence identity with 'Candidatus Phytoplasma ziziphi' (GenBank accession: CP025121). And tuf sequences of the strains showed 100% similarity with 'Ca. P. ziziphi' (CP025121). Interestingly, the virtual RFLP patterns derived from three 16Sr RNA gene sequences obtained in the study by iPhyClassifier (Zhao et al., 2009) were different from the reference patterns of all previously established 16Sr groups/subgroups. The most similar are the reference pattern of the 16Sr group VII, subgroup E (AY741531), with a similarity coefficient of 0.72, which is less than 0.85. These phytoplasma strains may represent a new 16Sr group. Phylogenetic analysis based on 16S rRNA genes using MEGA 7.0 by neighbor-joining (NJ) method with 1000 bootstrap value indicated that PeY-Heze strains clustered into one clade with the phytoplasma strains of 'Ca. P. ziziphi' with 68% bootstrap value. Although there are several reports available on 'Ca. P. solani' infecting peony in Shandong Province, China (Gao et al., 2013). To our knowledge, this is the first report of 'Ca. P. ziziphi'-related strains infecting peony in China. The findings in this study will be beneficial to the detection, quarantine, and prevention of peony yellows phytoplasmas in China.

Background: Paulownia witches’ broom disease (PaWB), caused by phytoplasmas, results in economic losses. Thymidine kinase (TDK) is a key enzyme in the deoxythymidine triphosphate (dTTP) synthesis pathway of phytoplasma, and dTTP serves as an essential precursor for DNA replication during cell proliferation, but its role in regulating phytoplasma proliferation and virulence remains unclear. Results: To characterize the role of TDK in phytoplasma proliferation and virulence, in this study, two PaWB phytoplasma tdk genotypes were identified via sampling in China, and tdk-1 was the most widely distributed. Phylogenetic analysis revealed that single-copied tdk exhibited higher phylogenetic resolution than 16S rRNA for phytoplasma molecular classification. In silico prediction showed that TDK functions as a thymidine kinase in these phytoplasmas. Furthermore, TDK protein expression was detected in PaWB-infected tissues. Immunofluorescence microscopy showed that TDK co-localized with phytoplasmas in sieve tube elements. Immunoelectron microscopy showed that TDK mainly localized in dividing phytoplasma cells. Absolute quantitative analysis revealed that tdk mRNA expression was positively associated with phytoplasma proliferation. Overexpressing tdk in Pseudomonas syringae pv. tomato ( Pst ) DC3000 significantly enhanced bacterial growth and increased its virulence in Arabidopsis . Conclusions: Overall, these results identify phytoplasma TDK as a critical proliferation-associated pathogenicity factor that promotes pathogen proliferation, providing a potential molecular target for controlling PaWB.