Cloning of Proteinase Inhibitor Gene StPI in Diploid Potato and Its Expression Analysis

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Agricultural Sciences in China 2007, 6(11): 1315-1321

ScienceDirect

November 2007

Cloning of Proteinase Inhibitor Gene StPI in Diploid Potato and Its Expression Analysis LI G u a n g - c u n 1 - 2 , JIN Li-pingl, XIE Kai-yunl, LI Yingl and QU Dong-yu1 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China Bio-Tech Center, Shandong Academy of Agricultural Sciences, Jinan 250100, P.R.China

Abstract A full-length cDNA of proteinase inhibitor gene with completed open reading frame of 116 amino acids was cloned from Ralstonia solanacearum (Rs) resistant potato leaves using the rapid amplification of cDNA ends (RACE) method and designated as StPI. BLAST search against NCBI showed that the StPI gene shared 89% identity with potato proteinase inhibitor I precursor in nucleotide and 74% in amino acid. Analysis of semi-quantitative RT-PCR indicated that this gene was induced by Rs as well as up-regulated by jasmonic acid (JA). The StPI gene expression reached the highest level during 6-12 h post Rs-inoculation or JA-treatment,and then leveled off. Moreover, this gene was strongly induced by JA

and its mRNA accumulation increased more quickly than that of Rs-inoculation. The StPI gene may play a role in potato resistance against Rs. The induction of StPI by Rs invasion may have a similar signal transduction pathway with JA treatment. Key words: potato bacterial wilt, RACE, StPI gene, full-length cDNA, gene expression

INTRODUCTION The cultivated potato (Solanum tuberosum L.) is one of the most important food crops, ranking at the fourth place in global food production. It is continually threatened by many pathogens and pests, which annually results in billions of US dollar losses. Bacterial wilt caused by Ralstonia solanacearum (Rs)is an important disease that spreads worldwide and infects hundreds of plant species, such as potato, tomato, banana, pepper and even trees. Moreover, high variability and diversity of Ralstonia solanacearum resulted in great losses of potato production (Grover et al. 2006; Castillo and Greenberg 2007). The bacterial wilt defense reaction in potato is a complicated continuous process that involves the expression of a battery of genes. But only

a few genes originated from potato were isolated and considered to play a role in Rs resistance, such as API (Feng et al. 2003). Proteinase inhibitors (PIS) are small proteins which are quite common in nature and also present in all life forms. PIS are found in storage organs, such as seeds and tubers, as well as in non-storage tissues, such as leaves, flowers, and roots (Brzin and Kidric 1995; de Leo et al. 2002; Sin and Chye 2004). Plant PIS are involved in plant defense and regulation of endogenous proteinases (Mosolov et al. 2001; Birk 2003). The defensive role of PIS is based on their inhibitory activities towards the proteinases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development (de Leo and Gallerani 2002), or interfering with important biochemical or physiological processes

This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence QU Dongyu, Tel: +86-10-68919805, Fax: +86-10-68975105, E-mail [email protected],net.cn

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of insects and other pathogens, such as the proteolytic activation of enzymes, molting of insects, or replication of viruses (Gutierrez-Campos et al. 1999). Since the first PI (trypsin inhibitor) was isolated from soybean, more than 495 inhibitors were identified in 129 different plants (de Leo et al. 2002). Jasmonic acid (JA) is an important signal element. It plays an important role in signal transduction as well as regulates plant proteinase inhibitor gene expression in pathogen defense (Orozco-Chrdenas and Ryan 2002; Chen et al. 2004; Halim et al. 2006). The EST fragment of StPZ gene was obtained through suppression subtractive hybridization (SSH) library construction and EST sequencing in the previous work. In the present study, RACE strategy was used to get the full-length cDNA of StPZ gene from Rs-resistant diploid potato genotypeED13. Moreover, the expression patterns of Rs and JA induction were also investigated by performing semi-quantitativeRT-PCR.

RNA extraction and ss-cDNA synthesis Total RNA was isolated from Rs-infected, JA- and water-treated potato leaves using TRIzol reagent (Invitrogen) and dissolved in DEPC-treated water. The quantity and quality of the total RNA was checked by the spectrophotometer (OD,/OD,,,) and formaldehydecontaining agarose gel electrophoresis. According to the manual of BD SMARTMRACE cDNA amplificationkit (Clontech),RACE-Ready cDNA was synthesized from RNA mixture of Rs-treated 6/12 hrs’ leaves using BD PowerScriptTMreverse transcriptase and 3’-CDS primer A (5’-AAGCAGTG GTATCAACGCAGAGTAC(T)3OVN-3’)provided in the RACE kit, Single strand cDNA (ss-cDNA) used for RT-PCR was individually synthesized from all treated samples in 20 pL reactions using Superscript I1 polymerase (Invitrogen) and oligo-dT( 18) primer (Shenggong Ltd., Shanghai, China) according to the manufacturer’s instructions.

MATERIALS AND METHODS Materialsand treatments The potato-infected ascendant Rs strain PO41 (Race 3) was kindly provided by Professor Feng Lanxiang, Plant Pathology Laboratory, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The potato genotype ED13 (progeny of 772102.37[E] x USW7589.2[D]) with high resistance against Rs strain PO41 was identified from the ED population provided by Professor Qu Dongyu (1996). The parent E of the ED population, originated from S. phureja and S.vemei, carries resistant genes against Rs. The potato plants (ED13) were planted in greenhouse with temperature of 18°C at night, 25°C during the day and with a 14h light period. Rs inoculation was carried out using a modified version “stem-bacterial co-culture method” (Li et al. 2006), and the samples were collected at 0, 6, 12, 24, 48, 72, and 96 h post Rs-inoculation. The JA-treated potato leaves were prepared by spraying 50 pnol L-l JA and harvested at 0, 3, 6, 12, 24, 36,48, and 72 h post JA-treatment. Control leaves were treated with water. Harvested leaves were frozen in liquid nitrogen and stored at -80°C for RNA extraction.

The forward gene specific primer (GSP-F: 5’-GGAGG GAAAGAGTATGCTCAAG’IT- 3’) was designed based on the 5’-end sequence of StPZ gene (a differentially expressed fragment in SSH library, EST129), and the 3’-RACE PCR was carried out according to the manual of S M A R T RACE cDNA amplificationkit using primer pairs of Universal Primer A (provided in the RACE kit) and GSP-F. PCR products were purified with the QIAquick@Spin PCR product purification kit (Qiagen) and cloned into pMD-18T vector (TaKaRa) and sequenced. The 3’-end sequence was assembled with 5’-end sequence of StPZ gene and the similarity search of the assembled full-length cDNA was performed against NCBI using BLAST program (BLASTX and BLASTN).

RT-PCR analysis To equal the starting amount of each sample, the fuststrand cDNAs were diluted 5-20 times (5-20 ng starting template) according to their PCR products amount of Actin gene with Actin specific primers (Actin-F: 5’-

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Cloning of Proteinase Inhibitor Gene StPI in Diploid Potato and Its Expression Analysis

GATGGTGTCAGCCACAC-3’;Actin-R: 5’-AlTCCAG CAGCTTCCATTCC-3’). According to the full-length cDNA sequence of StPZ gene, the gene specific primers (RT-F: 5’- TGCTTTCT TGCTTCTTGCATC-3’; RT-R: 5 -TTTAACAATGGC CTCCCAGTC-3’) were synthesized and used to analyze the StPZ gene expression by RT-PCR. To quantify original template quantities and maintain their initial differences of each analyzed sample, the PCR reaction was stopped as early as possible in order to get unsaturated PCR products accumulation. PCR reactions were subjected to 17-35 cycles at 94°C for 3 min; 94°C for 30 s, 54°C for 45 s and 72°C for 30 s. Five microliters PCR products of each sample was fractionated on 1.2% (w/v) agarose gel in tris-acetate EDTA buffer and stained with 0.5% (w/v) ethidium bromide. The expression level was estimated according to the intensity of ethidium-bromide-stained DNA bands.

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RESULTS RACE analysis The EST fragment (EST129 in SSH library) of StPZ gene was obtained previously. Results of bioinformatics analysis and BLAST search against NCBI showed that EST129 is the 5’-end of StPZ gene and includes signalpeptide, trans-membrane region and initiation coden ATG. In this study, only 3’-RACE amplification was carried out as the strategy of Fig. 1. Five microliters of 3’-RACE PCR products were separated on 1.2% agarose gel. The DNA band was specific and clear, and the size was about 550 bp (Fig.2).

Molecular characterizationof StPl gene The 3’-RACE sequence was assembled with the 5’ end

Region of overlap (100 bp) 1

-

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Region amplified by 3’-RACE

5’-

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Generalized first-strand cDNA

1

Fig. 1 RACE strategy for cloning the full-length cDNA of SfPI gene.

sequence of StPZ gene. To confirm that the 3’-RACE product was another fragment of StPZ gene, similarity searches of 3’-RACE sequence and 5’ end sequence of StPZ gene were carried out against the NCBI database. The results showed that they are different fragments of StPZ gene and can be assembled a full-length cDNA. The StPZ gene (GenBank accession No: DQ822994) contains an open reading frame (ORF) of 351 bp encoding 116 amino acids, 18 bp 5’-end un-translated region (UTR), complete poly(A) tail, and 3’-end untranslated sequence including putative polyadenylation signals AATAAAA (Rothnie 1996) located at 13 bp upstream of the poly(A) tail (Fig.3-A). Bioinformatics analysis showed that the StPZ gene contains classical protehase inhibitor domain, the signal-

Fig. 2 3‘-RACE results of StPZ cDNA.

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peptide and trans-membrane region. The StPZ gene shared 89% identity with potato proteinase inhibitor I precursor in nucleotide (accession No: DQ168314.1) and 74% in amino acid (Fig.3-B). BLAST results also suggested that the 5’-end of StPZ gene was conserved and the 3’-end was relatively variable.

Response of StPl gene to Rs and JA To identify if the StPZ gene was induced by Rs and JA, semi-quantitativeRT-PCR was used to analyze the StPZ gene expression. Accumulation of transcripts showed that StPZ gene was induced by Rs as well as up-regulated by JA. But they have different expression patterns. The mRNA accumulation of StPZ gene was increasing until 12 h post treatment, then leveled off. In contrast, this gene was strongly induced by JA. Its expression level increased faster and leveled off more quickly than that of Rs treatment (Fig.4-A, -B), whereas no obvious

A

1 70

136 20 1 267 333 409

changes in control (Fig.4-C), indicating that the StPZ gene maintains low level expression for participating plant basal metabolism in vivo in normal environment, and it is activated and shows expression increase when invaded by various pathogen or in abiotic-stress. It was concluded that this gene may play a role in potato resistance against Rs. Rs invasion may use the same component in signal transduction pathway or use similar pathway with JA-treatment.

DISCUSSION There are a number of methods for cloning full-length cDNAs. cDNA library screening is a widely used method and is preferred by many researchers for its reliability. But it also has many limitations, such as complicated procedure for hybridization, high false positive and low full-length cDNA clones. Rapid amplification of cDNA ends (RACE) is a simple cDNA

AMTAAAATAAAAGCM ATG GAG GGA AAG AGT ATG CTC AAG TTA TCT CAT GTG CM GCT TTC 7 T G CM M E G K S M L K L S H V I. A F L L

m GCA TCA c r r

in CAA TCA CTG ATG GCA AGA GAT TTG ATC ACT GAT GGC ATA GAA GTA CTG L A S L F Q S L M A K D L I S D G I E V L ATT CTG GAA AAT GAA ATC CAA GAT GTA TTG TGC CCA GGT AAG CAA TCA TGG CCT GAA CTT GTT I L E N E I Q D V L C P G K Q S W P E L V AAG CCA GCA GAA TAT CXX AAG M ATA ATT GAG AAG GAA AAT CCC ATA GCT CAT GAT ATT AGA

CAA Q GGG

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K P A E Y A K K I I E K E N P I A H D I R V 1TA TM CCT GGT ATG CTT AGG CCA TCT AAT TAT GTT TGT GGT AGA GTT TTT CTG GTT GTT GAC TGG 1, F P G M L R P S N Y V C G R V F L V V D W GAG CXX A T GTT AAA AIT ACT CCC ATA ATG GGT T A A T T A A T T A ~ A T G G G A A C A ~ A T G ~ A T G A A A A E A l V K I T P I M G * A M G T G G A G ~ r A C T M T A T A G ~ T ~ C A T A T A G T G T C I T C A T G T A C m A C T

496

B

SfpI 19

MEGKSMlklshvlaflllaslfqsl~~LISDGIEVLQI-LENEIQDVLCPGKQS~EL

195

MEGKSMLKLSHVLAFLLLASLFQ LWRDLISDGIEVLQ+ +EN+ + V CPGKQSWPEL PI-11

hlEGKSMLKLSHVLAFLLLASLFQPL~LISDGIEVLQLPVENDGEFVFCPGKQSWPEL

StPI 196

VGKPAEYAKKIIEKENPIAHDIRVLFPGMLRSNYVCGRVFLVVDWEAIVKITPIMG

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VGK A YAK++IEKEN I H++++LFPGM +P NYVCGRVFLVV+++ +V++TP MG PI-I61

VGKSAGYAKQVIEKENS1VHEVKLLFPGMPKPLNYVCGRVFLWNFKLVVQ~‘TPSMG

117

Fig. 3 A, nucleotide sequence and the deduced amino acid sequence of StPI gene. The nucleotide sequence is numbered on the left. The deduced amino acid sequence is shown underneath the correspondingnucleotide sequence. A putative polyadenylation signal is bolded. Untranslated regions are underlined. Stop code indicated with *. B, alignment of the deduced amino acid sequence of StPZ with potato proteinase inhibitor I precursor (accession No: AAZ94182.1).

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Cloning of Proteinase Inhibitor Gene StPZ in Diploid Potato and Its Expression Analysis

Fig. 4 RT-PCR analysis of transcript levels of StPI gene in potato leaves. cDNA was synthesized from total RNAs of different samples harvested at different time points. Gene specific primers of Actin gene were used to verify that the quantity of cDNA template of each sample was equal. Different time points are indicated on the top of the Fig. A, inoculated with Ralstonia solanacearum; B, treated with jasmonic acid; C, treated with water.

cloning strategy for its PCR reaction based on single strand cDNAs, which avoids the initial mRNA difference to be enlarged during the double strand cDNAs synthesis. Since the RACE technique was first reported (Frohman et al. 1988), it has been successfully used in many fields, for instance, identification of whole cDNAs, preparation of cDNA-library probes, cloning of flanking sequences of known gene fragments, and isolation of regulatory elements (Reddy et al. 2001; Lingle and Dyer 2001). The key factor of RACE is to design primers logically. High GC content (55-70%) and high Tm value (more than 70°C) are necessary for PCR specificity. Some other issues should also be noticed as well, conserved region is not proper choice for primer designing; selecting high-expressed tissue at appropriate timing point is important. If non-specific amplification still exists, the nested gene-specific primers should be used

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for further PCR amplification. Since the size of the StPI gene is less than 1 kb and its 5’-end sequence has been obtained in previous study, only the 3’-RACE was performed and the whole cDNA sequence of the StPI gene was obtained easily. Plants protect themselves against microbial pathogens through various different responses. The most efficient way is to strengthen their defense arsenal. Proteinase inhibitors are natural, defense-related proteins often present in seeds and induced in certain plant tissues by pathogens. These proteinase inhibitor genes, especially inhibitor genes of plant origin, will be activatedin general and contribute to plant resistance when invaded by various pathogens or in abiotic stress (Koiwa et al. 1997; Fan and Wu 2005). In addition, proteinase inhibitors have also been implicated to play a role in natural defense towards fungal infections (Soares-Costa et al. 2002) and modulate programmed cell death (PCD) in plants (Solomon et al. 1999), etc. Furthermore, the gene transfomation of trypsin inhibitor gene could improve the broad spectrum resistance to pathogens and insects in tobacco (Hilder et al. 1987). The StPI gene in this study has high identity with potato proteinase inhibitor I precursor, indicating that it may be a member of potato proteinase inhibitor gene family and play an important role in defense towards Rs. To date, three signalingmolecules, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), are known to play key roles in various aspects of plant defense. These three signaling molecules are involved in two major pathogen defense signaling pathways: an SA-dependent pathway and a JA/ET-dependent pathway. These. pathways do not function independently, but rather influence each other through a complex network of regulatory interactions (Bostock 2005). JA plays an important role in basal resistance reaction against P. infestans in potato (Halim et al. 2006). Unfortunately, knowledge on SA-regulated resistance in potato is limited. Recently, two independent pathways related with proteinase inhibitor gene expression in potato leaves were characterized,one involving JA (Dong 1998), and one involving ABA (Fan and Wu 2005). In some cases, they co-regulate gene expression together, which depends on a certain gene and reaction (Bostock 2005; Chao et al. 1999). In this study, the StPI gene was induced by Rs as well as up-regulated by JA.

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Interestingly, the StPZ gene was strongly induced by JA, which may be caused by the special JA-regulation fashion or JA treatment method of directly spraying JA on potato leaves in this study. The result indicated that Rs invasion may use a similar signal transduction pathway with JA-treatment in regulating StPZ gene expression. The StPZ gene function and the role of SA and ABA in regulating StPZ gene expression need to be further studied.

Acknowledgements We thank Professor Feng Lanxiang in the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences for kindly providing Ralstonia solanacearum strain P041. This research was funded by the Opening Laboratory of Vegetable Genetics and Physiology of Ministry of Agriculture, China.

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Cloning of Proteinase Inhibitor Gene StPI in Diploid Potato and Its Expression Analysis

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