The Role of TaRanGAP2 in Wheat Resistance to Puccinia triticina Infection

doi:10.7668/hbnxb.20192497

Abstract

Abstract:

The aim to study the role of TaRanGAP2 in HR induction of wheat resistance to Puccinia triticina,so as to lay a foundation for elucidating the mechanism of Puccinia triticina resistance and breeding for disease resistance in wheat at the molecular level.The wheat near-isogenic line TcLr26 and its recurrent parent Tatcher(Tc)were used to form incompatabile(TcLr26×260)and compatabile combination(Tc×260)with Puccinia triticina race 260,respectively.Bioinformatics analysis showed that the total length of CDS of TaRanGAP2 was 1 665 bp,encoding 554 amino acids.Analysis of its conserved domains revealed that the protein encoded by TaRanGAP2 gene belonged to the Ran GTPase family;RT-qPCR was used to detect the expression of TaRanGAP2 in the two combinations.It was found that the expression of TaRanGAP2 was significantly up-regulated in the incompatible combination;the subcellular localization detection of TaRanGAP2 by tobacco transient transformation system showed that TaRanGAP2 was localized in the cytoplasm;Virus induced gene silencing(VIGS)technology were used to silence TaRanGAP2 gene in TcLr26.After inoculation with Puccinia triticina race 260,it was found that HR area of TaRanGAP2 gene-silencing plants was significantly increased,and the number of HMCs were also significantly increased compared with the unsilenced plants control plants.These results proved that TaRanGAP2 played a positive regulatory role in the induction of HR in wheat resistance to Puccinia triticina.

Key words: Wheat, Puccinia triticina, TaRanGAP2, Hypersentive reaction

SONG Shanshan, ZHANG Xinting, WANG Qi, LU Yuanjing, HOU Chunyan, WANG Dongmei. The Role of TaRanGAP2 in Wheat Resistance to Puccinia triticina Infection[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 158-164. doi: 10.7668/hbnxb.20192497.

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Figures/Tables 8

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	引物用途 Primer application
qRT-F	ACGCGAACTACATCCCAGAC	RT-qPCR
qRT-R	CTTCGTCCTCGTCATCAGGG
GAPDH-F	CTGCATCATACGATGACATC	RT-qPCR内参基因
GAPDH-R	TGTCACCGACAAAGTCAGTG
TaRanGAP2-GFP-F	CGTCGACATGGCTTCAGCGGCGCAAGATTTC	亚细胞定位试验
TaRanGAP2-GFP-R	CACTAGTGTCCTCCTCCGCCTCCACCTTCAG
VIGS-TaRanGAP2-F	GTGCGGCCGCTCCCGCAAGTATGGCCTTTTG	VIGS试验
VIGS-TaRanGAP2-R	CAGCGGCCGCGCAGTCGTCACTGGACCTTTTTTC

Tab.2 System of VIGS assay

试验设计 Experiment design	反应混合液Reaction mixture
试验组Test group	α、β、BSMV:TaRanGAP2各2 μL+19.5 μL FES
对照组Control group	α、β、BSMV:00各2 μL+19.5 μL FES
指示组Indicating group	α、β、BSMV:PDS各2 μL+19.5 μL FES

Fig.1 Analysis of the conserved domain and species phylogenetic tree of TaRanGAP2
A.Conserved domain:Green area is the conserved domain of WPP;Red area is the LRR leucine repeat sequence.B.TaRanGAP2 is in the red box.

Fig.2 Effect of Puccinia triticina inoculation on TaRanGAP2 gene expression
RT-qPCR values are means±s of three independent experiments data,SPSS software is used to analyze the significance of the difference.The mean value indicated by the same letter has no significant difference(P>0.05).The same as Fig.4,6.

Fig.3 Subcellular localization of TaRanGAP2

Fig.4 Leaf phenotype and silencing efficiency detection
A.Observation of leaf phenotype 13 days after virus transfection;B.The detection of TaRanGAP2 gene silencing efficiency by RT-qPCR.

Fig.5 Fluorescence staining of VIGS silent leaves inoculated with Puccinia triticina
Number of HMC observed by ultraviolet excitation;HR area observed by blue-light excitation.

Fig.6 The statistics of HMC number and HR area of single infection site
A.Statistics of the number of HMCs at a single infection site after inoculation; B.Statistics of HR area of single infection site after inoculation.

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