Establishment of Analog-sensitive Protein Kinase Research System in Plant Pathogenic Fungi

doi:10.13560/j.cnki.biotech.bull.1985.2021-1602

Abstract

Abstract:

Common gene knockout techniques cannot be applied to the study of some specific kinases. In order to provide new ideas and methods for the study of protein kinases，an analog-sensitive protein kinase system based on artificial modification of protein kinases was established in plant pathogenic fungi. The “gatekeeper” residues of protein kinase were predicted via web technology，and the analog-sensitive（as）mutants gpmk1-as and pmk1-as were obtained by vector complement method. The function of analog-sensitive protein kinase and its sensitivity to ATP analogue inhibitor 1-NM-PP1 were detected. The results showed that the “gatekeeper” residues of Gpmk1 and Pmk1 were Q103 and Q104，respectively，which were very conserved in multiple representative fungi. The colony growth rate of the analog-sensitive mutant gpmk1-as was consistent with that of the wild type PH-1，both faster than that of the Gpmk1 knockout mutant. pmk1-as produced appressorium being same with that of the wild type Guy11，but the Pmk1 knockout mutant could not do so. With 5 μmol/L 1-NM-PP1，the colony growth rate of Gpmk1-as decreased，but the growth of PH-1 was not affected. With 10 μmol/L 1-NM-PP，pmk1-as could not form appressorium，but Guy11 formed mature black appressorium. The results showed that both Gpmk1 and Pmk1 analog-sensitive protein kinases could perform normal protein functions，but were very sensitive to ATP analogue inhibitor 1-NM-PP1.

Key words: plant pathogenic fungi, protein kinase, artificial modification, chemo-genetical technique, analog-sensitive

SUN Zhong-juan, LIU Qian-qian, GUO Yu-qian, WANG Guang-hui, WANG Chen-fang. Establishment of Analog-sensitive Protein Kinase Research System in Plant Pathogenic Fungi[J]. Biotechnology Bulletin, 2022, 38(11): 49-57.

Figures/Tables 9

Table 1 Primer sequences used in this study

引物名称Primer	引物序列Sequence（5'-3'）
GPMK1-as/1F	AGGGAACAAAAGCTGGGTACCGGGGTATGGT- GGTCAAGGTTATG
GPMK1-as/2R	ACAGTGGCGTTTCGTACTCCGATCAGATAGAC
GPMK1-as/3F	GTCTATCTGATCGGAGTACGAAACGCCACTGT
GPMK1-as/4R	GATTTCAGTAACGTTAAGTGGATCCATGGTGG- CAAGTAAAGGAATGAG
GPMK1-as/5F	CTGTATCCGTAGATGGGAGGTA
GPMK1-as/6F	GACCGTGCTTGCTGTATCCT
GPMK1-as/7F	GACCGAACGACCGATTGACT
GPMK1-as/8F	CAACCTCCTCCTCAACGCCAACT
GPMK1-as/9F	ACCCATACCTTGAGCCTTACC
PMK1-as/1F	AGGGAACAAAAGCTGGGTACCGCGTCAAGCC- ATCGCGTTATCT
PMK1-as/2R	GAGTGTTCAGCAGCAACGGACCCCGATCAGG- TACACCTCGTT
PMK1-as/3F	TTCAACGAGGTGTACCTGATCGGGGTCCGTTG- CTGCTGAACACTCC
PMK1-as/4R	GATTTCAGTAACGTTAAGTGGATCCGGCACAG- CCAATCCACGAATACACA
PMK1-as/5F	ATTCTCGTTGGTCCATCCGTC
PMK1-as/6F	TCCACCAAGCAACTCTTCGGG
PMK1-as/7F	TCAAGGCAATGCACTCGGCAAAC
PMK1-as/8F	GTACCATGACCCCGATGATGAACC
PMK1-as/9F	CGGCACTCACAATTAAGCCGTTG
GPMK1-5F	CAACTGTGATCTCAAGGTCTGCG
GPMK1-6R	TGCTTCAGAGCTTCCTCCACAG
PMK1-5F	CGAAACGCCGTCTACTGTACTTG
PMK1-6R	CAGTACTGGGGAGAACATCGTG

Fig. 1 Construction of pKNT-GPMK1-as vector

Fig.2 Multiple sequence alignments of Gpmk1 in representative fungi（A）and 3D structure of Gpmk1（B）

Fig. 3 “Gatekeeper” residues of some essential protein kinases

Fig.4 PCR detection of gpmk1-as（A）and pmk1-as（B）transformants M：DNA marker；P：positive control；N：negative control

Fig.5 Cultures of the strains PH-1，gpmk1 and gpmk1-as grown on PDA plate for 2 d（A）and colony dia-meters of the indicated strains（B） Different letters indicate significant differences based on ANOVA analysis followed by Duncan’s multiple range test（P=0.05）

Fig. 6 Appressorium formation of strains Guy11，pmk1 and pmk1-as on hydrophobic slides（Bar=50 μm）

Fig.7 Cultures of the strain PH-1，gpmk1 and gpmk1-as grown on PDA plate for 2 d after 1-NM-PP1 treatm-ent（A）and colony diameters of the indicated str-ains（B） Significant differences based on t test（P=0.05）

Fig.8 Appressorium formation of strain Guy11，pmk1 and pmk1-as on hydrophobic slides after 1-NM-PP1 treatment（Bar=50 μm）

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