Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 65-73.doi: 10.3864/j.issn.0578-1752.2020.01.006

• PLANT PROTECTION • Previous Articles     Next Articles

Functional Analysis of Gene ShARPC5 Involved in Tomato Resistance to Powdery Mildew

ChanJing FENG,GuangZheng SUN,Yang WANG(),Qing MA()   

  1. College of Plant Protection, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
  • Received:2019-07-10 Accepted:2019-08-21 Online:2020-01-01 Published:2020-01-19
  • Contact: Yang WANG,Qing MA E-mail:wangyang2006@nwsuaf.edu.cn;maqing@nwsuaf.edu.cn

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Abstract:

【Objective】Powdery mildew is an important disease in tomato production, and seriously affects the yield of tomato. Genome sequences of Solanum lycopersicum provide valuable information resources for disease-resistant gene searching. The actin- related protein 2 and 3 complex (ARP2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. In this study, tomato ARPC5, encoding a subunit protein of the ARP2/3 complex, was cloned and identified for disease resistance. The results will lay a foundation for upgrading tomato genomic information, further studying the resistance mechanism and molecular breeding.【Method】ShARPC5 was annotated from the genomic databases and cloned from tomato LA1777 (S. habrochaites). DNAMAN 6.0 was used for multiple sequence alignment and MEGA 6.0 was used for constructing phylogenetic tree. The prediction of protein subcellular localization was performed using ProtComp v. 9.0. The qRT-PCR was used to compare the expression of ARPC5 in the high-sensitive variety Moneymaker (MM) and high-resistant variety LA1777 induced by Oidium neolycopersici (On-Lz). The correlation between On-Lz infection and ARPC5 expression was analyzed. The function of ShARPC5 involving in tomato resistance to On-Lz was further verified by the technology of virus-induced gene silencing (VIGS). The phenotypic changes of ShARPC5-silenced and wild-type lines were observed. Hypersensitive response (HR) and H2O2 production were detected by trypan blue and DAB staining, respectively, and the expression of some marker genes related to plant disease resistance was assayed in ShARPC5-silenced plants. Additionally, genetic transformation of Arabidopsis thaliana was conducted by Agrobacterium-mediated floral-dip method. The phenotypic changes of transgenic and wild-type lines were observed, and the number of conidia per lesion was also counted.【Result】The S. habrochaites ARPC5 was identified and characterized. ShARPC5 encodes a 132-amino-acid protein possessing a conserved P16-Arc domain. Compared with the compatible interaction between tomato MM (Moneymaker) and On-Lz, the expression of ShARPC5 was significantly up-regulated in incompatible interaction between tomato LA1777 and On-Lz, especially at 18 hpi. Silencing of ShARPC5 in tomato could increase the susceptibility to the powdery mildew pathogen On-Lz. The expression of PR1b1, a maker gene related to signal regulation, was significantly down-regulated after silencing of ShARPC5. The histological observation showed that the induction of hypersensitive cell death and the generation of reactive oxygen were reduced in silenced-ShARPC5 tomato plants compared with wild types. Transient over-expression of ShARPC5 in tobacco could rapidly produce necrotic spots. Conversely, over-expression of ShARPC5 in A. thaliana, followed by inoculation with On-Lz, showed enhanced resistance.【Conclusion】ShARPC5 is an important gene which can reduce the incidence of tomato powdery mildew, and has a great application value in the mechanism study of tomato resistance to powdery mildew. At the same time, it can be used as a candidate gene for molecular breeding of tomato powdery mildew resistance.

Key words: tomato powdery mildew, resistance, actin cytoskeleton, ARP2/3 complex, ARPC5

Table 1

Genes and primers in qRT-PCR reaction"

基因
Gene		 引物序列Primer sequence
正向引物Forward primer sequence (5′-3′) 反向引物Reverse primer sequence (5′-3′)
ShARPC5/SlARPC5 CGAAGGCATAATCACAAGA CAGCAAGACAACGCAGTA
PR1b1 CATCCCGAGCACAAAAC TGAAGTCACCACCACCCT
Glucanase A CTTTTACTTGTTGGGCTTCT ACTTCCTTTGAGGGCATT
Chitinase 3 ACGCCATCCCCTAAAGA TGGACCCATCCCACATT
GAPDH (Control) CTGGTGCTGACTTCGTTGTTG GCTCTGGCTTGTATTCATTCTCG
AtARPC5 CGGAATGCTCAATGCTCT CGGTCCCCAGTAGAAAGTC
UBQ10 (Control) AGAAGTTCAATGTTTCGTTTCATGTAA TTACGAATCCGAGGGAGCCATTG

Fig. 1

Sequence analysis of tomato actin related protein 2 and 3 (ARP2/3) complex subunit 5 (ShARPC5)"

Fig. 2

ARPC5 expression in tomato leaves analyzed by qRT-PCR"

Fig. 3

Effect of silencing of ShARPC5 in tomato LA1777 plants on the infection ability of On-Lz strain"

Fig. 4

Effect of silencing of ShARPC5 on tomato defense responses"

Fig. 5

Hypersensitive necrosis induced by ShARPC5 in N. benthamiana a: pGR106:GFP (CK); b: pGR106:ShARPC5; c: Buffer; d: pGR106:GFP:BAX; e: pGR106:ShARPC5:BAX; f: Buffer-BAX"

Fig. 6

Resistance of over-expression of ShARPC5 in A. thaliana against On-Lz"

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