Abstract: 【Objective】The objective of this study is to clone a gene PsPik1 encoding type III PtdIns 4-kinase from Puccinia striiformis f. sp. tritici (Pst) and characterize its function during Pst growth and development. 【Method】The full-length cDNA of PsPik1 was obtained using RT-PCR. Bioinformatic methods were used to analyze molecular properties of PsPik1. Quantitative real-time PCR (qRT-PCR) was applied to examine the expression profiles of PsPik1 at twelve different development stages. The transcript level of PsPik1 was calculated by the 2^-△△CT method with the EF1 gene of Pst as endogenous reference for normalization. BSMV-HIGS was employed to analyze the function of PsPik1 by knocking down its expression. Selected PsPik1 gene fragment was amplified by PCR from Pst cDNA using primers with restriction enzymes Not I and Pac I sites. Amplicon was ligated into the BSMV γ vector generating BSMV:γ:PsPik1-as. The native BSMV:γ:0-as and BSMV:γ:TaPDS-as were used as negative and positive control, respectively. After inoculation with BSMV at the two-leaf stage, wheat seedlings were maintained in a growth chamber and examined for symptoms at regular intervals. After 10-day post virus inoculation, the fourth leaves of inoculated wheat seedlings in the PsPik1 silencing group were inoculated with urediospores of Pst race CYR32. The fourth leaves were collected at 24, 48 and 120 hours post inoculation (hpi) for histological observation and detection of silencing efficiency. Stained leaf segments were examined with an Olympus BX-51 microscope for the numbers of hyphal branches and haustorial mother cells and lengths of infection hyphae at 48 hpi and for colony size at 120 hpi. The incidence of stripe rust was observed and recorded with camera at 12 days post inoculation (dpi).【Result】 Based on the bioinformatic analysis and RT-PCR, the full-length cDNA of PsPik1 was cloned. The open reading frame (ORF) of PsPik1 was 4 485 bp in length, encoding 1 494 amino acids containing lipid kinase unique domain (LKU), NCS1 binding site, Hom2 domain and PI3K/PI4K superfamily catalytic domain. Phylogenetic analysis indicated that PsPik1 was clustered into PI4KIIIβ group and more closely related to Pik1 from Puccinia group than to Pik1 from ascomyceteous fungi and PI4KIIIβ from animals and plants. qRT-PCR assays revealed that PsPik1 transcripts was up-regulated within 6-24 hpi and peaked at 12 hpi. PsPik1 expression at 6, 12, 18 and 24 hpi were 2.66-, 7.04-, 3.42- and 2.96-folds as high as that of resting urediospores. However, expression of PsPik1 was down-regulated between 36 hpi and 5 dpi with the lowest expression at 5 dpi when it was 6.67-fold lower than that of resting urediospores. From 7 to 11 dpi, PsPik1 expression was gradually increased and the amount of transcripts was as high as that of resting urediospores at 11 dpi. To determine cytological changes associated with fungal growth on plants, silenced for PsPik1 wheat leaves inoculated with race CYR32 were examined microscopically. The hypha length and the number of hypha branches of Pst at 48 hpi and the colony size per infection site at 120 hpi in BSMV:γ:PsPik1-as infected wheat leaves were much shorter than that in BSMV:γ:0-as infected plants (control). PsPik1 transcript level exhibited an average of 49%, 49% and 69% expression in BSMV:γ:PsPik1-as infected plants as high as that in control plants.【Conclusion】PsPik1 may play an important role in the regulation of plant penetration and infectious growth in Pst.

Key words: Puccinia striiformis f. sp. tritici, type III PtdIns 4-kinase, qRT-PCR, BSMV-HIGS