Differential expression of Rs-eng-1b in two populations of Radopholus similis (Tylenchida:Pratylecnchidae) and its relationship to pathogenicity Chao Zhang.Hui Xie.Chun-Ling Xu Xi Cheng-Ke-Mei Li.Yu Li 825202omc161ac302 Abstract We constructed a ssh (suppression sub was approximately 2.7 times as much as the expre tractive hybridization)library based on two popula tions (Rs-C and Rs-P)of Radopholus similis from different host plants and exhibiting differences in path paradisiaca and Anthu from the R-Cpopulationw ogenicity on Musa nression differ es amo four different develor plants.In order to screen the clones with ment stages The order of Rs-eng-lb relativ significant ex ssion differences from the SSH li ssion abundance from high to lo was females ry.a total of 2400 clones was randomly selected iuveniles males and e We furthe used RNAi to and reverse northern blotting was performed on them test whether Rs Out of the 2,400 clones,89 clones showed significant sponsible for nathogenicity which was the first rnai expression differences.Out of sequencing these 89 work about Rs-eng-1b.The RNAi results showed that clones distinct sequences from 87 clones were Rs-eng-b exnression had a nositive correlation to obtained.Aligning the 87 distinct sequences against pathogenicity of the population.The longer the the non-redundant nucleotide database (nr)in nCBl RNAi treatment,the less pathogenic the nematode we found that five sequences were highly conserved population was.Non-endogenous gfp dsRNA had no with Rs-eng-/6.Two of five sequences with lengths of significant influence on the expression of Rs-eng-/b 467 base pairs (bp)(GW395922)and 742 bp and pathogenicity of R.similis Rs-C population.In (GW395923)were further emploved to perform 5 conclusion,all our evidence indicated Rs-eng-/b RACE-PCR and 3'RACE-PCR,respectively might be a crucial pathogenicity-related gene in R. Subsequently,the complete length of Rs-eng-/b similis (EU414839)was obtained (1,427 bp).Our qPCR re sult showed that expression of Rs-eng-Ib in the pop Keywords Banana burrowing nematode.SSH ulation Rs-C with high pathogenicity on host plants B-1.4-endoglucanase.Real-time PCRRNAi Introduction ratory of Plant Nematology and Research Cente The banana burrowing nematode,Radopholus similis is one of the most damaging pests of bananas and 510642.People's Republic of China severely harms pepper,omamental plants,and many -mail:xichui@scau.edu.cn other agronomic and horticultural crops.R.similis is Springe
Differential expression of Rs-eng-1b in two populations of Radopholus similis (Tylenchida: Pratylecnchidae) and its relationship to pathogenicity Chao Zhang & Hui Xie & Chun-Ling Xu & Xi Cheng & Ke-Mei Li & Yu Li Accepted: 25 May 2012 / Published online: 16 June 2012 # KNPV 2012 Abstract We constructed a SSH (suppression subtractive hybridization) library based on two populations (Rs-C and Rs-P) of Radopholus similis from different host plants and exhibiting differences in pathogenicity on Musa paradisiaca and Anthurium andraeanum plants. In order to screen the clones with significant expression differences from the SSH library, a total of 2,400 clones was randomly selected and reverse northern blotting was performed on them. Out of the 2,400 clones, 89 clones showed significant expression differences. Out of sequencing these 89 clones, distinct sequences from 87 clones were obtained. Aligning the 87 distinct sequences against the non-redundant nucleotide database (nr) in NCBI, we found that five sequences were highly conserved with Rs-eng-1b. Two of five sequences with lengths of 467 base pairs (bp) (GW395922) and 742 bp (GW395923) were further employed to perform 5′ RACE-PCR and 3′ RACE-PCR, respectively. Subsequently, the complete length of Rs-eng-1b (EU414839) was obtained (1,427 bp). Our qPCR result showed that expression of Rs-eng-1b in the population Rs-C with high pathogenicity on host plants was approximately 2.7 times as much as the expression of Rs-eng-1b in the population Rs-P with low pathogenicity on host plants. Furthermore, the gene Rs-eng-1b from the Rs-C population also showed expression differences amongst four different development stages. The order of Rs-eng-1b relative expression abundance from high to low was females, juveniles, males, and eggs. We further used RNAi to test whether Rs-eng-1b of Rs-C population was responsible for pathogenicity which was the first RNAi work about Rs-eng-1b. The RNAi results showed that Rs-eng-1b expression had a positive correlation to pathogenicity of the population. The longer the RNAi treatment, the less pathogenic the nematode population was. Non-endogenous gfp dsRNA had no significant influence on the expression of Rs-eng-1b and pathogenicity of R. similis Rs-C population. In conclusion, all our evidence indicated Rs-eng-1b might be a crucial pathogenicity-related gene in R. similis. Keywords Banana burrowing nematode . SSH . β-1 . 4-endoglucanase . Real-time PCR . RNAi Introduction The banana burrowing nematode, Radopholus similis is one of the most damaging pests of bananas and severely harms pepper, ornamental plants, and many other agronomic and horticultural crops. R. similis is Eur J Plant Pathol (2012) 133:899–910 DOI 10.1007/s10658-012-0015-4 C. Zhang : H. Xie (*) : C.-L. Xu : X. Cheng : K.-M. Li : Y. Li Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, College of Natural Resource and Environment, South China Agricultural University, Guangzhou 510642, People’s Republic of China e-mail: xiehui@scau.edu.cn
900 Eur J Plant Pathol (2012)133:899-910 known to attack over 250 plant species (O'Bannon populations of Globodera pallida and identified a cel- 1977).R.similis is a migratory endoparasite that lulase and an important pathogenicity factor invades the roots and feeds on the cytoplasm of Meanwhile,several parasite-related genes from cortex cells.As a result,the roots will blacken and Meloidogyne incognita were detected by SSH (Huang die which results in a reduction of plant growth et al.2004).Although mining differentially expressed and development.This destruction of crops leads genes in PPNs is not very difficult,it is crucial to to severe economic losses and consequently,many understand gene function involved in pathogenesis and countries have entered it on the list of quarantine regulation pattems.RNA interference (RNAi)is an plant pests (Cotton and Van Riel 1993;Smith and RNA-dependent gene silencing process in which the Charles 1998). guide strand(small interference RNA,siRNA)from Nematicides have been used as one of many inte short double-stranded RNA (dsRNA)molecules are grated approaches to control plant-parasitic nematodes incorporated into the RNA-induced silencing complex (PPNs).As concems have arisen over the environmen (RISC)to bind to specific mRNA molecules (target tal implications associated with over-using of some mRNA).siRNAs prevent target mRNAs from being translated into protein by either degrac ding target n use mRN om completely trans it yield loss ha Ihus,a ating the mRN. to seek additional app for PPNs contro n recent year molecular bio ing approa trol hav et a we ting diff ath 2000.Ma app che s to i 2004: PCR (R supp iRNA displa (RSDD p y app ole K Sher SSH has the d tiall 1999)SSH ad 1002. sitivity low of fals 2002 tal.2007 d infection in the plan ince more than 100 differ one ssH (vor t al B-1.4-endoglucanase (EGases)also known as cel 1997).Grenier et al.(2002) d SSH to and ie differentially pressed genes betw en two A classified as a fifth glycosy Springer
known to attack over 250 plant species (O'Bannon 1977). R. similis is a migratory endoparasite that invades the roots and feeds on the cytoplasm of cortex cells. As a result, the roots will blacken and die which results in a reduction of plant growth and development. This destruction of crops leads to severe economic losses and consequently, many countries have entered it on the list of quarantine plant pests (Cotton and Van Riel 1993; Smith and Charles 1998). Nematicides have been used as one of many integrated approaches to control plant-parasitic nematodes (PPNs). As concerns have arisen over the environmental implications associated with over-using of some nematicides, many nematicides have been decreased in use. As a result, shortcomings in our ability to successfully limit yield loss have occurred. Thus, an effort to seek additional approaches for PPNs control is imperative. In recent years, molecular biology and genetic engineering approaches to solve the problem of PPNs control have been developed quickly. In order to have more appropriate biological controls, understanding the molecular mechanisms involved in pathogenicity by mining pathogenicity genes has become extremely important (Chen et al. 2005). It is an effective method to screen for pathogenicity genes by comparing gene expression profiles between different populations of a species exhibiting different levels of pathogenicity on host species. There are a series of approaches to identify differentially expressed genes, such as mRNA differential display reverse transcription PCR, representational difference analysis (RDA), suppression subtractive hybridization (SSH), and reciprocal subtraction differential RNA display (RSDD). Of those approaches, SSH has been extensively applied in molecular genetics and molecular biology (Kuang and Ashorn 1993; Tchernitsa et al. 1999; Shen and Liu 2004). SSH has super sensitivity to recognize the differentially expressed genes in low expression abundance (Diatchenko et al. 1999). SSH advantages include high sensitivity, low occurrence of false positives, high efficiency, and low cost. Commonly, SSH is better than other approaches to detect differentially expressed genes, since more than 100 differentially expressed genes can be enriched in one SSH (von Stein et al. 1997). Grenier et al. (2002) used SSH to investigate differentially expressed genes between two different populations of Globodera pallida and identified a cellulase and an important pathogenicity factor. Meanwhile, several parasite-related genes from Meloidogyne incognita were detected by SSH (Huang et al. 2004). Although mining differentially expressed genes in PPNs is not very difficult, it is crucial to understand gene function involved in pathogenesis and regulation patterns. RNA interference (RNAi) is an RNA-dependent gene silencing process in which the guide strand (small interference RNA, siRNA) from short double-stranded RNA (dsRNA) molecules are incorporated into the RNA-induced silencing complex (RISC) to bind to specific mRNA molecules (target mRNA). siRNAs prevent target mRNAs from being translated into protein by either degrading target mRNAs or inhibiting ribosomes from completely translating the mRNA. RNAi has been found in many eukaryotes including animals and plants. RNAi was initially discovered and developed to application on potato (Kawchuk et al. 1991) and Caenorhabditus elegans (Guo and Kemphues 1995; Fire et al. 1998). RNAi can be used as a simple and effective alternative geneknockout tool to obtain function-less or function-loss mutants, because of its high sequence-specificity and effective interference activity. Meanwhile, with its simple operation, short cycle and low cost, it has become an extremely important tool and most popular research topic in the fields of gene identification, genetic analysis, gene function, gene therapy and genomics (Barstead 2001; Kamath et al. 2000; Maine 2001; Cheng et al. 2003; Rangasamy et al. 2004; Tijsterman et al. 2004). Although it is difficult to study gene function of PPNs by constructing mutants because PPNs are obligatory parasites and do not grow in artificial culture medium; with the advantage above, RNAi could be used for screening mutants, identifying gene function, or control of PPNs (Bakhetia et al. 2007). However, little research has been carried out on the pathogenicity genes of PPNs, and their functions; and, so far, research has also concentrated on the sedentary endoparasitism nematodes, such as Meloidogyne sp., Heterodera sp. and Globodera sp. (Goddijn et al. 1993; Brindley et al. 1997; Vercauteren et al. 2002; Shingles et al. 2007). The molecular mechanism of parasitic-related-gene-mediated infection in the plant host-parasite interaction is still unclear. β-1, 4-endoglucanase (EGases), also known as cellulase, functions in degrading plant cellulose and is classified as a fifth glycosyl hydrolase family 900 Eur J Plant Pathol (2012) 133:899–910
Eur J Plant Pathol (2012)133:899-910 901 Oniacgeman et al 2008)EGcne tanster con Nematodes 2007 ed RNAi to silence EGases in lod Two populations of R similis:Rs-C and Rs-P,which ath d Philoe odes with silen ntly les Rs. Rs.E sbeen wide th。 the ole in tw arg ned to pop natodes.The ltured on ex nm ion of p studied by of 6 cm at 25C in al (2008).and m seful in eidentified The four EGase Nematode extraction es in R.similis had been cloned by Haegeman et al.(2008).Their res The carrot callus was mashed with a blender The only focused on tisst ion and tim mashed solution was filtered through combined sieves (Hae n et al 2008)Currently t ie no bort or with aperture of 0.147 mm and 0.026 mm.Nematodes whether the function of EGases is involved in pathoge were collected from the 0.026 mm ap re si nicity of r.similis on plant Roots of anthurium were cut into I cm frag ent n this study.a differential ge ession libran These fra nts were mashed in a blender and furthe was constructed by SSH.based on tw onulation of filtered by nested sie R similis from different and different hosts des wer om the EGases in R.similis (Rs- 0.026mm eve.This first nematode collec ened from a ssh library. In tion was labelled as NI.Meanwhile.the 0.147 m ne function.a series of experiments including pore sieve subiected to the baermann funnel sepa RNAi.real-time PCR (aPCR).carrot callus culture ing the nematodes number was labelled as n2 The and artificial infection,were performed to investigate total nematodes in roots were calculated as the sum of the relationship between the expression of Rs-eng-/b NI and N2 Nematodes in soil were isolated from and pathogenicity of R.similis on plant. 200 ml of mixed soil from the host plant pot using a Baermann funnel method Extraction from sieve or Baermann funnel were adjusted to 10 ml of nematode Materials and methods suspension.and I ml of the suspension was pipetted into a glass dish with a diameter of 6 cm.and the Plant materials number of nematodes were counted under the stereo microscone while the number of females males and Anthurium,Anthurium andraeanum plants were iuveniles were counted respectively.The same work bought from the Flowers and plants research center was done 10 times until all nematode suspensions had Guangzhou,Guangdong.The roots of anthurium seed- e o been counted.Total nematode lings were washed with sterile water and nematodes collected on nested 0.147 mm and 0.026 mm pore (NI N2)and soil. sieves.Baermann funnels (Viglierchio and Schmitt 1983)were used to separate nematodes from the pre RNA extraction cipitation and microscope inspection was employed.If there were nematodes in roots.the roots were treated About 20.000 mixed-stage nematodes from each pop- to remove nematodes (Tsang et al.2004).All non- ulation separated from carrot disks were respectively contaminated seedlings were grown in sterilized soil collected in an Eppendorf tube and washed with dieth medium for 15 days for later use. ypyrocarbonate (DEPC)water three times.Cleaned Springe
(Haegeman et al. 2008). EGases was first thought to originate through horizontal gene transfer (HGT) (Jones et al. 2005). Chen et al. (2005) and Bakhetia et al. (2007) employed RNAi to silence EGases in Globodera rostochiensis and Heterodera glycines, respectively. Their investigation showed that the nematodes with silenced EGases had significantly less pathogenicity on their host plants. It has been widely accepted that EGases plays a key role in pathogenicity in the two nematodes. Therefore, EGases is viewed as a prospective target gene that could be developed to provide resistance to the nematodes. The first high throughput molecular characterization of R. similis was studied by Jacob et al. (2008), and much useful information was discovered; some genes involved in parasitism, including EGases were identified. The four EGases genes in R. similis had been cloned by Haegeman et al. (2008). Their research only focused on tissue-expression and time-expression (Haegeman et al. 2008). Currently, there is no report on whether the function of EGases is involved in pathogenicity of R. similis on plant. In this study, a differential gene expression library was constructed by SSH, based on two populations of R. similis from different areas and different hosts. EGases in R. similis (Rs-eng-1b) was selectively screened from a SSH library. In order to clarify the gene function, a series of experiments including RNAi, real-time PCR (qPCR), carrot callus culture, and artificial infection, were performed to investigate the relationship between the expression of Rs-eng-1b and pathogenicity of R. similis on plant. Materials and methods Plant materials Anthurium, Anthurium andraeanum plants were bought from the Flowers and Plants Research Center, Guangzhou, Guangdong. The roots of anthurium seedlings were washed with sterile water and nematodes collected on nested 0.147 mm and 0.026 mm pore sieves. Baermann funnels (Viglierchio and Schmitt 1983) were used to separate nematodes from the precipitation and microscope inspection was employed. If there were nematodes in roots, the roots were treated to remove nematodes (Tsang et al. 2004). All noncontaminated seedlings were grown in sterilized soil medium for 15 days for later use. Nematodes Two populations of R. similis: Rs-C and Rs-P, which were collected from roots of ornamental plants Calathea makoyana and Philodendron cv Green Emerald, respectively, and the internal transcribed spacer (ITS) regions of Rs-C and Rs-P were sequenced. The different pathogenicity of the two nematode populations was certified on Musa paradisiaca by a member of our group (personal communication). These populations were cultured on excised carrot (Daucus carota) disks in Petri dishes with a diameter of 6 cm at 25 °C in incubator (Fallas and Sarah 1994). Nematode extraction The carrot callus was mashed with a blender. The mashed solution was filtered through combined sieves with aperture of 0.147 mm and 0.026 mm. Nematodes were collected from the 0.026 mm aperture sieve. Roots of anthurium were cut into 1 cm fragments. These fragments were mashed in a blender and further filtered by nested sieves with apertures 0.147 mm and 0.026 mm. Nematodes were collected from the 0.026 mm aperture sieve. This first nematode collection was labelled as N1. Meanwhile, the 0.147 mm pore sieve subjected to the Baermann funnel separating the nematodes number was labelled as N2. The total nematodes in roots were calculated as the sum of N1 and N2. Nematodes in soil were isolated from 200 ml of mixed soil from the host plant pot using a Baermann funnel method. Extraction from sieve or Baermann funnel were adjusted to 10 ml of nematode suspension, and 1 ml of the suspension was pipetted into a glass dish with a diameter of 6 cm, and the number of nematodes were counted under the stereomicroscope, while the number of females, males and juveniles were counted respectively. The same work was done 10 times until all nematode suspensions had been counted. Total nematode population size was calculated as the sum of nematodes isolated from roots (N1 + N2) and soil. RNA extraction About 20,000 mixed-stage nematodes from each population separated from carrot disks were respectively collected in an Eppendorf tube and washed with diethypyrocarbonate (DEPC) water three times. Cleaned Eur J Plant Pathol (2012) 133:899–910 901
902 Eur J Plant Pathol (2012)133:899-910 nematodes were ground in liquid nitrogen.Total RNA non-redundant protein database(nr)and a non- was extracted from the nematodes using TRIZOL redundant nucleotide database (nt)in NCBL following manufacturer instructions (Invitrogen)and further treated with DNase I(Promega)for 15 min at Obtaining complete sequence of Rs-eng-/b 37 C.The RNA was verified by 1.0 agarose gel electrophoresis and was stored-80C for later use. The fragment candidate of Rs-eng-Ib was screened Only 500 mixed stages nematodes separated from out of the library with alignment analysis.To obtain carrot disks were treated by RNAi.Therefore,a the complete sequence of Rs-eng-/b,3RACE primers MicroElute total RNA kit (OMEGA)was used to (NEST-S1 and NEST-S2)(Table 1)and 5'RACE extract the total RNA according to the kit operation primers (NEST-Aland NEST-A2)(Table 1)were protocol. designed to amplify the 3'end and 5'end of Rs-eng /b using a SMART RACE cDNA amplification kit SSH library (Clontech),respectively.Finally,we spliced three frag ments of Rs-eng-Ib(5'end,middle fragment,and 3 A SMARTer PCR DNA Synthesis Kit (Clontech) end)into the complete sequence of Rs-eng-/6.Two tran tota RNA ron A-C an specific primers (cds-F and cds-R)(Table 1)from to cDN ording t complet sequences of Rs-eng-Ib were anu cture designed to form the complete sequence of Rs-eng-/b went using a PCR-S on ki Expression analysis of Rs-eng-Ib and qPCR secondary I amplifie produ nt pGEM-T easy ega and qPCR was used to asse the var on in the e expres tran plet Escherich a coli JM1 eng-Ib be etween Rs-Ca -P ne cells.Final single were ran omly selecte ato ent for later PCR detecting and sequencing. eggs carrot callus Reverse Northern blotting juvenile Total RNAs from Rs-Cand Rs-P nematode population ively wer R h synthesize P fro PCR de populati prime DNA la samples as three biol dete on th cDNA ed CR-R by ano-Dr DNA abe pro kit and P om SSH d Rs-p nd c Sequencing and alignment analysis d in trinlic CFX-96 (Bio-Rad)qPCR machir SYBR GO After hybridization the results of the hybridized sis lus kit (TOYOBO) the nale analysed by UV transilluminator (Alpha and 60C for 30eA0 verse orthern blotting signals wer elected for cing.After CEX-96 ted ct valu and adapter ea uences, alig extrap olated relative levels of PCR products from Springer
nematodes were ground in liquid nitrogen. Total RNA was extracted from the nematodes using TRIZOL following manufacturer instructions (Invitrogen) and further treated with DNase I (Promega) for 15 min at 37 °C. The RNA was verified by 1.0 % agarose gel electrophoresis and was stored −80 °C for later use. Only 500 mixed stages nematodes separated from carrot disks were treated by RNAi. Therefore, a MicroElute total RNA kit (OMEGA) was used to extract the total RNA according to the kit operation protocol. SSH library A SMARTer PCR cDNA Synthesis Kit (Clontech) was used to transcribe total RNAs from Rs-C and Rs-P nematode populations into cDNA according to the manufacturer’s protocol. Resulting cDNAs underwent Suppression Subtractive Hybridization (SSH) using a PCR-Select cDNA subtraction kit (Clontech). The secondary PCR amplified product was cloned into the vector pGEM-T easy (Promega) and was then transformed into complete Escherichia coli JM109 cells. Finally, single clones were randomly selected for later PCR detecting and sequencing. Reverse Northern blotting Total RNAs from Rs-C and Rs-P nematode populations were transcribed into cDNA according to operation instruction of a ReverTra Ace qPCR RT kit (TOYOBO). To synthesize Probe-C and Probe-P from Rs-C and Rs-P nematode populations, a DIG high prime DNA labelling and detection starter kit I (Roche) was used to label the cDNA templates. The concentration of probes was quantified by a Nano-Drop spectrophotometer. According to operation protocol of DIG high prime DNA labelling and detection starter kit I (Roche), the PCR products from SSH were hybridized with Probe-C and Probe-P. Sequencing and alignment analysis After hybridization, the results of the hybridized signals were analysed by UV transilluminator (Alpha Innotech). Blots that had two-fold differences in reverse northern blotting signals were selected for sequencing. After removing vector sequences and adapter sequences, sequences were aligned against a non-redundant protein database (nr) and a nonredundant nucleotide database (nt) in NCBI. Obtaining complete sequence of Rs-eng-1b The fragment candidate of Rs-eng-1b was screened out of the library with alignment analysis. To obtain the complete sequence of Rs-eng-1b, 3′RACE primers (NEST-S1 and NEST-S2) (Table 1) and 5′ RACE primers (NEST-A1and NEST-A2) (Table 1) were designed to amplify the 3′ end and 5′ end of Rs-eng- 1b using a SMART RACE cDNA amplification kit (Clontech), respectively. Finally, we spliced three fragments of Rs-eng-1b (5′ end, middle fragment, and 3′ end) into the complete sequence of Rs-eng-1b. Two specific primers (cds-F and cds-R) (Table 1) from spliced complete sequences of Rs-eng-1b were designed to form the complete sequence of Rs-eng-1b. Expression analysis of Rs-eng-1b and qPCR qPCR was used to assess the variation in the expression levels of Rs-eng-1b between Rs-C and Rs-P nematode populations, and among different development stages of Rs-C: females, males, juveniles and eggs. RsC and Rs-P were isolated from infected carrot callus for total RNA extraction. One hundred females, males, juveniles and eggs respectively were used for RNA extraction. The RNA was then quantified and qualified using a Nano-drop spectrophotometer, and then stored at −80 °C for further analysis. All the RNA used for qPCR was prepared from three different samples as three biological replicates. Based on the complete sequence of Rs-eng-1b, specific primers qPCR-F and qPCR-R (Table 1) were designed to represent Rs-eng-1b expression. According to the method described by Jacob et al. (2007), the primers Actin-F and Actin-R were synthesized (Table 1) to amplify the reference gene, β-actin. qPCRs were performed on mixed life stages of Rs-C and Rs-P and females, males, juveniles, and eggs of RsC, and reactions were performed in triplicate on CFX-96 (Bio-Rad) qPCR machine, using SYBR Green qPCR Master Mix-plus kit (TOYOBO) according to the manufacture’s protocol with the following reaction conditions: 95 °C for 15 s and 60 °C for 30 s (40 cycles). Initial data analysis was carried out using the Bio-Rad CFX-96 manager software, which created Ct values and extrapolated relative levels of PCR products from 902 Eur J Plant Pathol (2012) 133:899–910
Eur J Plant Pathol (2012)133:899-910 903 Table 1 Primers used for RACE of cndoglucanase Rs it-/6 Primer Sequence Source hain rea Actin- S-GAAAGAGGGCCGGAAGAG-3 Jacob et al.(2007 Actin-R 5-AGATCGTCCGCGACATAAAG-3 Jacob et al.(2007) on in Rs lation NEST S 5-ACGAGACCTACAATGAGC-3 NEST S2 5-AGCCTGCCCGTGTTCGTGAC-3 NEST Al S-GGCAGGTACTCGGTCACG-3 (ds)RNA of Rs-eng-Ib NEST A2 5-CATTGTAGGTCTCGTACC-3 OPCR-E 5-AATCTCTTACGTGAACTGGGC-3 OPCR-R SGGTCGCTCCAGATTTAGTCG-3 cds-E 5.TCCGCTTTCACCGCTTTCA-3 cds-R SCAGACATTCAGCATCCA3 T7S S-TAATACGACTCACTATAGGGGCTGTI A TCATTGTAG-3 NGGTGGGCTCATTGTAG-3 G-T7S G-A 5-CGATGCGGTTCACCAGGGTGTCG-3 G-T7A Gs 5CACAAGTTCAGCGTGTCCGGCG-3 standard curves.Melt curves were done routinely.and nd G)(Table)o gp)was generated with desig necific nrimer this allowed the possibility of hoth contamination and primer dimers to be discounted.Actin was used as a cloning vector PYL 322-d1-GFPn supplied by QL positive control in all experiments.All experiments Zhu,College of Life-Science,South China Agricultural were performed in triplicate. University. Synthesis of Rs-eng-Ib dsRNA Rs-eng-/b's RNAi treatment and silence detection A fragment of about 450 bp from the ORF of Rs-eng Five hundred nematodes from the Rs-C population Ib was cloned into the vector PMD18-T (TAKARA) cultivated on carrot callus were collected and trans The constructed vector was further confirmed by se ferred in an Eppendorf tube,treated with DEPC quencing.Based on the fragment,specific primers water and soaked in 50 ul rs-eng-lb dsRNa solution (T7S.A T7A.and S)(Table 1)with a T7 promoter (2 ug/ul)at room temperature for 12 h.24 h.36 h were designed to amplify the sense and anti-sense and 48 h,respectively.Non-endogenous gfp dsRNA product.Sense and antisense rNA were transcribed solution (2 ug/ul)was used as a control.The using T7 transcription kit(TOYOBO)according to the treatment times used for the control were same as manufacturer's instructions.Sense and antisense Rs-eng-/b dsRNAs.Meanwhile,untreated nematodes transcripts were annealed for 30 min at 37 C and were used as a blank control.Treated nematodes were analyzed by agarose gel electrophoresis.The dsRNA cleaned with DEPC water three times and the total RNA was purified by equal amount of LiCl(3 mol/l) was then extracted.aPCR was used to analyze transcript ovemight and washed by 70%ethylalcohol three times suppression of Rs-eng-/b in the nematodes after the finally stored at-80C for later use.Non-endogenous RNAi treatments.All experiments were performed control dsRNA (125 bp)(green fouorescent protein three times. Springe
standard curves. Melt curves were done routinely, and this allowed the possibility of both contamination and primer dimers to be discounted. Actin was used as a positive control in all experiments. All experiments were performed in triplicate. Synthesis of Rs-eng-1b dsRNA A fragment of about 450 bp from the ORF of Rs-eng- 1b was cloned into the vector PMD18-T (TAKARA). The constructed vector was further confirmed by sequencing. Based on the fragment, specific primers (T7S, A, T7A, and S) (Table 1) with a T7 promoter were designed to amplify the sense and anti-sense product. Sense and antisense RNA were transcribed using T7 transcription kit (TOYOBO) according to the manufacturer’s instructions. Sense and antisense transcripts were annealed for 30 min at 37 °C and analyzed by agarose gel electrophoresis. The dsRNA was purified by equal amount of LiCl (3 mol/l) overnight and washed by 70 % ethylalcohol three times, finally stored at −80 °C for later use. Non-endogenous control dsRNA (125 bp) (green fouorescent protein gene, gfp) was generated with designed specific primers (G-T7S, G-A, G-T7A, and G-S) (Table 1) from the cloning vector PYL 322-d1-GFPn supplied by QL Zhu, College of Life-Science, South China Agricultural University. Rs-eng-1b’s RNAi treatment and silence detection Five hundred nematodes from the Rs-C population cultivated on carrot callus were collected and transferred in an Eppendorf tube, treated with DEPC water, and soaked in 50 μl Rs-eng-1b dsRNA solution (2 μg/μl) at room temperature for 12 h, 24 h, 36 h and 48 h, respectively. Non-endogenous gfp dsRNA solution (2 μg/μl) was used as a control. The treatment times used for the control were same as Rs-eng-1b dsRNAs. Meanwhile, untreated nematodes were used as a blank control. Treated nematodes were cleaned with DEPC water three times and the total RNA was then extracted. qPCR was used to analyze transcript suppression of Rs-eng-1b in the nematodes after the RNAi treatments. All experiments were performed three times. Table 1 Primers used for RACE of endoglucanase Rs-eng-1b gene sequence, quantitative polymerase chain reaction (qPCR) for Rs-eng-1b expression in Rs-C population and Rs-P population and different development stage of Radopholus similis, and to generate double-stranded (ds) RNA of Rs-eng-1b and non-endogenous gfp dsRNA control Primer Sequence Source Actin-F 5′-GAAAGAGGGCCGGAAGAG-3′ Jacob et al. (2007) Actin-R 5′-AGATCGTCCGCGACATAAAG-3′ Jacob et al. (2007) NEST S1 5′-ACGAGACCTACAATGAGC-3′ NEST S2 5′-AGCCTGCCCGTGTTCGTGAC-3′ NEST A1 5′-GGCAGGTACTCGGTCACG-3′ NEST A2 5′-CATTGTAGGTCTCGTACC-3′ qPCR-F 5′-AATCTCTTACGTGAACTGGGC-3′ qPCR-R 5′-GGTCGCTCCAGATTTAGTCG-3′ cds-F 5′-TCCGCTTTCACCGCTTTCA-3′ cds-R 5′-CAGACATTCAGCATCCA-3′ T7S 5′-TAATACGACTCACTATAGGGGCTGTT CTGGTCGCAATG-3′ A 5′-CAGAGGTGGGCTCATTGTAG-3′ T7A 5′-TAATACGACTCACTATAGGGCAG AGGTGGGCTCATTGTAG-3′ S 5′-GCTGTTCTGGTCGCAATG-3′ G-T7S 5′-GGATCCTAATACGACTCACTATAGGG CACAAGTTCAGCGTGTCCGGCG-3′ G-A 5′-CGATGCGGTTCACCAGGGTGTCG-3′ G-T7A 5′-GGATCCTAATACGACTCACTATAGGG CGATGCGGTTCACCAGGGTGTCG-3′ G-S 5′-CACAAGTTCAGCGTGTCCGGCG-3′ Eur J Plant Pathol (2012) 133:899–910 903