Virus induced gene silencing of AtCDC5
Research article
Virus induced gene silencing of AtCDC5results in accelerated
cell death in Arabidopsis leaves
Zhiqiang Lin a ,Kangquan Yin a ,Xiaoxiao Wang a ,Meihua Liu a ,Zhangliang Chen a ,b ,
Hongya Gu a ,b ,Li-Jia Qu a ,b ,*
a
National Laboratory for Protein Engineering and Plant Genetic Engineering,Peking-Yale Joint Research Center for Plant Molecular Genetics and
AgroBiotechnology,College of Life Sciences,Peking University,Yuan Ming Yuan Rd.5,Beijing 100871,People’s Republic of China
b
The National Plant Gene Research Center (Beijing),Beijing 100101,People’s Republic of China
Received 21July 2006;accepted 19December 2006
Available online 27December 2006
Abstract
CDC5,a Myb-related protein,is reported to be essential for the G 2phase of cell cycle in yeast and animals,but little is known about its function in plants.In this study,Arabidopsis thaliana CDC5(AtCDC5)is found to be nuclear localized,and the C-terminus of this protein is of transcriptional activation activity in yeast.By taking advantage of the virus induced gene silencing (VIGS)technique,we analyzed the phenotypes of the plants in which AtCDC5is speci?cally silenced.The AtCDC5VIGS plants died before bolting,in which accelerated cell death was detected.Further analysis showed that the transcripts of AtSPT and SAG13,but not SAG12,accumulated in these AtCDC5VIGS plants,suggesting that the accelerated cell death is different from that occurred during leaf senescence.Furthermore,silencing of AtCDC5by VIGS in either wild-type,npr1or nahG plants all induces cell death,suggesting that SA is not crucial for the AtCDC5-associated cell death.ó2006Elsevier Masson SAS.All rights reserved.
Keywords:Virus-induced gene silencing (VIGS);AtCDC5;Transcriptional factor;Cell death;Salicylic acid
1.Introduction
Myb-related protein CDC5is functionally and structurally conserved in eukaryotes [26].The ?rst CDC5gene,cdc5tfrom Schizosaccharomyces pombe ,was isolated as an essential gene for G 2phase,and null cdc5tmutant spores could germi-nate,but died as elongated cells [25].Although human CDC5
was found to regulate the mitotic entry probably by acting as a transcription factor [3,17],studies in ?ssion yeast and human cells showed that CDC5protein is a crucial component of spli-ceosome [1,23],and is essential for mRNA splicing [6,35].For example,loss of CDC5/Cef1p function in yeast results in cell cycle defects by in?uencing the splicing of TUB1mRNA [7].In plants,cell cycle plays an essential role in development,and many cell cycle regulators have been studied at the whole plant level [15].For instance,over-expression of non-degrad-able cyclinB1in tobacco leads to growth retardation,and even post-germination death of seedlings in some extreme cases.In these plants,cytokinesis was found impaired and endomito-sis occurred,suggesting that disturbed cell cycle is responsible for the growth defects [36].In another case,mis-expressing ICK/KRP1,a possible inhibitor of CDKA;1,in Arabidopsis trichomes inhibited endoreduplication and induced cell death,which in turn could be rescued by mis-expression of CYCD3;1in trichomes [33].However,the mechanism of cell death
Abbreviations:CaMV ,cauli?ower mosaic virus;DAI,days after in?ltra-tion;DIC,differential interference contrast;ORF,open reading frame;PCD,programmed cell death;RT-PCR,reverse transcription-polymerase chain reaction;SA,salicylic acid;TRV ,tobacco rattle virus;VIGS,virus induced gene silencing.
*Corresponding author.National Laboratory for Protein Engineering and Plant Genetic Engineering,Peking-Yale Joint Research Center for Plant Molecular Genetics and AgroBiotechnology,College of Life Sciences,Peking University,Yuan Ming Yuan Rd.5,Beijing 100871,People’s Republic of China.Tel.:t861062753018;fax:t861062753339.
E-mail address:qulj@https://www.sodocs.net/doc/182003011.html, (L.-J.Qu).0981-9428/$-see front matter ó2006Elsevier Masson SAS.All rights reserved.
doi:10.1016/j.plaphy.2006.12.003
Plant Physiology and Biochemistry 45(2007)87e
94
https://www.sodocs.net/doc/182003011.html,/locate/plaphy
happens in these transgenic plants is not clear.In plants,pro-grammed cell death(PCD)is an important process for proper development and defense response,including treachery ele-ments formation,leaf senescence,deletion of suspensor and aleurone cells,as well as HR response[27].Several marker genes accumulated during leaf senescence,such as AtSPT, SAG12and SAG13,have been identi?ed[10,22].Meanwhile, salicylic acid is known to be a phytohormone that triggers pro-grammed cell death(PCD)[16,31],and several mutants exhib-iting spontaneously SA-dependent cell death,such as acd6-1, acd11,ssi1and lsd6,have been characterized[5,12,32,34].To clarify whether cell death caused by cell cycle disturbance is a programmed cell death and whether it is SA-dependent will facilitate to understand the roles of cell cycle genes in plant development.
In this study,we found that AtCDC5was a nuclear localized protein,and that its C-terminus was of transcriptional activation activity in yeast.In order to investigate its function in vivo,we knocked down the expression of AtCDC5in wild-type Arabi-dopsis plants by using virus induced gene silencing(VIGS). The AtCDC5VIGS plants we generated,in which AtCDC5 was speci?cally silenced as shown by northern blots as well as RT-PCR,died before bolting.Staining assay with trypan blue detected accelerated cell death in the rosette leaves of these AtCDC5VIGS plants.Northern blots showed that AtSPT and SAG13,but not SAG12,accumulated in these plants.Further-more,silencing of AtCDC5by VIGS in npr-1and nahG plants resulted in similar phenotype with those AtCDC5VIGS plants in the wild-type background,suggesting that SA level is not crucial for the cell death caused by silencing AtCDC5.
2.Results
2.1.Characterization of AtCDC5as a
transcription factor
Based on the bioinformatics analysis,AtCDC5is a member of Arabidopsis MYB-related transcription factor family[13]. The cDNA of AtCDC5gene was cloned in a genome-wide ORFeome cloning of Arabidopsis transcription factors [9,13].To test whether AtCDC5is localized to nuclei,we generated a GFP-AtCDC5fusion protein construct in which transient gene expression was driven by the strong CaMV35S promoter,and bombarded onion epidermal cells.As shown in Fig.1A,GFP-AtCDC5fusion protein was found exclusively localized in nuclear,as visualized by?uorescence microscopic and differential interference contrast(DIC)images(Fig.1A), suggesting that the AtCDC5protein is targeted to nuclei. Taking into consideration that AtCDC5is of DNA binding activity in vitro[14],it is reasonable to propose that AtCDC5 serves as a transcription factor.
To determine whether AtCDC5is involved in transcription regulation,we fused the entire coding sequence(aa1e844) and C-terminal region(aa335e844)of AtCDC5with the GAL4DNA-binding domain respectively(see Section4),and co-transformed them with the reporter vector[37].As shown in Fig.1B,the C-terminal region rather than the entire AtCDC5protein signi?cantly activated the LacZ reporter gene expression in yeast cells,suggesting that AtCDC5may serve as a positive regulator of transcription.This result also implies that the correct con?guration of the N-terminal region(mainly DNA binding domain)may be critical for the transactivation activity.
2.2.Generation of AtCDC5VIGS plants
We requested the mutant for AtCDC5from NASC,and found no obvious phenotype for heterozytous plants but recovered no homozygous plants in the T4generation offsprings,suggesting that loss-of-function mutant of AtCDC5is lethal(data not shown),probably because that the CDC5protein is structurally and functionally conserved in eukaryotes[26].Therefore,in order to investigate the physiological role of AtCDC5,we adop-ted the virus induced gene silencing(VIGS)system to knock down the expression level of AtCDC5[20,21].To guarantee the speci?city,a BLASTN search was conducted with the partial sequence of AtCDC5(500e2535,excluding the conserved MYB domain)as the probe and a365-bp fragment(637e 1001)was selected due to its low sequence similarity(nucleo-tide sequence identity<5%,E value?1.9)with other genes in Arabidopsis genome.This fragment was then ampli?ed by PCR and cloned into pTRV2,recombinant designated pTRV2-AtCDC5(Fig.2A)(see Section4).
To carry out VIGS,the third and fourth true leaves of3-week-old Arabidopsis seedlings were in?ltrated with a mixture of Agrobacterium cultures containing pTRV1and pTRV2-AtCDC5constructs,whereas mixture of Agrobacterium cultures containing pTRV1and pTRV2was used as a negative control, and a mixture of Agrobacterium cultures containing pTRV1 and pTRV2-PDS was used as a positive control.At15days after in?ltration(DAI),the negative control plants appeared similar to the wild-type plants,whereas8out of9positive controls ex-hibited the albino phenotype[20](Fig.2B,C),suggesting that this VIGS system works well in the Arabidopsis plants in our lab.The development of those control plants was not affected and in?orescences were well developed21DAI(Fig.2B,C). As shown in Fig.2B,17out of21plants in?ltrated with pTRV2-AtCDC5were found having distorted rosette leaves 15days after in?ltration.Lesions gradually formed on the ro-sette leaves of AtCDC5VIGS plants,but no in?orescences were generated.At21DAI,all of these17AtCDC5VIGS plants died without bolting(Fig.2C).Trypan blue staining assay showed that cell death occurred in the leaves of the AtCDC5 VIGS plant(Fig.2D)but not in that of pTRV2in?ltrated plant (Fig.2D),suggesting accelerated cell death happened in AtCDC5VIGS plants during their development.For the other 4AtCDC5VIGS plants,they also showed accelerated death phe-notype soon after bolting(data not shown).
2.3.AtCDC5is speci?cally silenced in
AtCDC5VIGS plants
To clarify whether the accelerated death phenotype in the AtCDC5VIGS plants was due to silencing of AtCDC5, northern blots were carried out with the RNA from AtCDC5
88Z.Lin et al./Plant Physiology and Biochemistry45(2007)87e94
VIGS plants.For the Northen blot and RT-PCR assays,the RNA sample of each type of plants was extracted and pooled from 10independent plants with similar phenotypes.As shown in Fig.3A,the transcripts of AtCDC5were greatly re-duced in the AtCDC5VIGS plants.Meanwhile,the expression of RAN1,a copper-exporting ATPase encoding gene that showed highest sequence similarity to the AtCDC5VIGS frag-ment (637e 1001)in the BLASTN search,was not affected in AtCDC5VIGS plants (Fig.3B).These results suggest that the expression of AtCDC5was indeed speci?cally silenced in AtCDC5VIGS plants and that silencing of AtCDC5is likely responsible for the accelerated death phenotype in leaves.2.4.The expression of two PCD marker genes is dramatically up-regulated in AtCDC5VIGS plants
In plants,old leaf cells undergo PCD,which is also known as leaf senescence [27].To determine whether the accelerated
cell death occurred in the leaves of AtCDC5VIGS plants is associated with the cell death during the leaf senescence,we carried out northern blots to monitor the expression level of three genes,SAG12,SAG13[22]and AtSPT [10],which were accumulated during leaf senescence.The RNA gel blots showed that the expression level of SAG13and AtSPT was dramatically up-regulated,but that of SAG12was slightly down-regulated (Fig.4),suggesting that the cell death trig-gered by virus induced silencing of AtCDC5is different from cell death occurred during leaf senescence.2.5.Silencing of AtCDC5in npr1and nahG plants also induces accelerated cell death
In the mutant acd11that exhibited SA-dependent cell death,the expression level of SAG13was found up-regulated,whereas that of SAG12was not affected,and the accelerated cell death in acd11leaves was believed to be
different
Fig.1.Subcellular localization of AtCDC5and trans-activation activity assay.(A)Subcellular localization of AtCDC5.The images were inspected by a ?uorescence microscope (images shown as the left panel)and a differential-interference-contrast microscope (images shown as the right panel).The nuclear were indicated by arrows in the DIC pictures.Bars indicate 50m m.(B)Trans-activation activity assay.Full length and partial coding sequence of AtCDC5were respectively cloned into pYF503and fused in frame with the GAL4DNA binding domain.The numbers in the brackets indicate the corresponding amino acid sites of AtCDC5protein.pYF503derivates were transformed into yeast containing a pG221reporter vector,and b -galactosidase ?lter assays were carried out by using X-gal as a substrate.For every transformation,three independent transformants were shown.pYF504,which harbors the full-length GAL4gene,was used as a positive control.
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from that during leaf senescence [5].Based on the similar expression pattern of the SAG12and SAG13,it would be inter-esting to clarify whether the accelerated cell death occurred in acd11shares anything in common with that found in AtCDC5VIGS plants.To answer this question,we chose two SA-related mutants,npr1that is defective in SA signal transduction [8]and nahG that defective in endogeneous SA accumulation [11],to do VIGS with AtCDC5probe in order to examine whether the cell death found in the AtCDC5VIGS plants is SA-dependent.As shown in Fig.5B,D,11out of 12npr1plants in?ltrated by pTRV2-AtCDC5,died 19DAI,whereas 11out of the 15nahG plants in?ltrated by pTRV2-AtCDC5died 16DAI.These results suggest that the cell death caused by AtCDC5silencing is different from that occurred in acd11,and that SA level is not crucial for the AtCDC5-associated cell
death.
Fig.2.Phenotype of AtCDC5VIGS plants in wild type background.(A)Schematic representation of the pTRV2,pTRV2-AtCDC5and pTRV2-PDS constructs.AtCDC5VIGS fragment was cloned into the multiple cloning sites (MCS)of pTRV2.Both the pTRV2and the pTRV2-PDS constructs were previously described [20].(B)Wild-type plants,15days after in?ltration with Agrobacterium .There was no obvious phenotype for the plants infected with recombinant pTRV2alone.Plants infected with TRV carrying the pTRV2-AtCDC5formed distorted rosette leaves (indicated by arrows).Plants infected with TRV carrying the pTRV2-PDS showed photo-bleaching phenotype due to the inhibition of carotenoid biosynthesis [20].(C)Wild-type plants,21days after in?ltration with Agrobacterium .In-?orescence already formed in the plants infected by pTRV2alone or by pTRV2-PDS .Most part of the plant infected by pTRV2-AtCDC5died at this time;though ?ower buds can be seen (indicated by arrow),it can not bolt,and totally died in the following 3e 5days.(D)Partial of the seventh leaves of the Agrobacterium infected plants 18days after in?ltration,stained by trypan blue.Dead cells were detected in the seventh leave of AtCDC5VIGS plant,but not in those of the TRV infected plant.Bar:1mm.
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3.Discussion
Members of Myb-related CDC5protein family are structur-ally and functionally conserved in eukaryotes [26],and they have been reported to be essential for pre-mRNA splicing both in yeast and human [6,23],and their N-termini have DNA bind-ing activity [14,17,25].However,the function of CDC5in plants is yet to be illustrated.The fact that AtCDC5shares high sequence similarity with other MYB transcription factors and that AtCDC5is of DNA binding activity in vitro [14]raised the possibility that AtCDC5may serve as a transcription factor.In this study,we found that AtCDC5was nuclear localized (Fig.1A)and its C-terminal region had transcription activity in yeast (Fig.1B),suggesting that AtCDC5may serve as a positive regulator of transcription.However,the entire AtCDC5protein did not have transcriptional activation activity in our
trans-activation activity assay (Fig.1B).Similar phenomenon has been found in other MYB proteins.For example,LAF1,an Arabidopsis R2R3-MYB transcription factor involved in phytochrome A signaling,is acting as a transcriptional activator,but the entire LAF1protein has much lower transcriptional ac-tivation activity than its C-terminus [2].Meanwhile,both At-MYB21and AtMYB59are reported to have less trans-activation activity for the entire proteins than their C-termini [18].It is reasonable to assume that binding DNA at the N-ter-minal region,therefore forming a correct con?guration at the N-terminal region,might be critical for the transactivation activ-ity of these proteins.
It has been reported that yeast cdc5tgene functions at G 2phase,and its null mutant yeast cells are inviable [25].In our study,when AtCDC5was speci?cally silenced in Arabidopsis with the help of VIGS (Fig.3),the plants displayed accelerated cell death phenotype (Fig.2D).The structural and functional conservativeness among CDC5proteins from different species,together with the fact that AtCDC5has similar expression pattern with CDC2a [14],suggests a role of AtCDC5as a cell cycle regulator.It has been reported that cell cycle distortion caused by mal-function of cell cycle regulators can induce cell death.For instance,over-expressing non-destructive cyclinB1in tobacco or mis-expressing KRP1in Arabidopsis trichomes could all lead to cell death [33,36].Moreover,cell death in the plants mis-expressing KRP1even exhibited some characteristics of PCD,such as nuclear destruction [33].There-fore,it is reasonable to assume that the accelerated cell death occurred in AtCDC5VIGS plants is a result of cell cycle distor-tion caused by mis-expression of cell cycle regulator gene AtCDC5.The fact that two PCD marker genes (AtSPT and SAG13)were dramatically up-regulated in AtCDC5VIGS plants (Fig.4)suggests that the cell death found in the AtCDC5silencing plants is likely a kind of PCD.
However,SAG12which accumulates during leaf senes-cence [22]was slightly down-regulated in AtCDC5VIGS plants (Fig.4),suggesting that the cell death caused by AtCDC5silencing is not the same as the typical PCD during leaf senescence.Similar results was found in the acd11mutant which displays spontaneously cell death phenotype [5].In acd11,the expression of SAG13is up-regulated whereas SAG12is not affected,and the double mutant of acd11/nahG shows no accelerated cell death phenotype,suggesting that the cell death in acd11is SA-dependent [5].Different with acd11,silencing of AtCDC5both in the wild-type and in the nahG plants induces cell death (Fig.5),suggesting that SA level is not crucial for the AtCDC5-associated cell death.4.Methods 4.1.Plant materials
Arabidopsis thaliana (ecotype Columbia-0)plants were grown under long-day conditions (16h light/8h dark).The mutants npr1and nahG were kindly provided by Dr.Shuhua Yang (China Agriculture
University).
Fig.3.AtCDC5was speci?cally silenced in AtCDC5VIGS plants.(A)RNA gel blot analysis of AtCDC5in pTRV2infected plants and pTRV2-AtCDC5infected plants.Equal loading of the gel was con?rmed by EtBr staining (bot-tom panel).(B)RT-PCR analysis of RAN1expression in pTRV2infected plants and pTRV2-AtCDC5infected plants.The experiments were repeated for three times and the cycle number was
30.
Fig.4.The expression of PCD marker genes in AtCDC5VIGS plants.RNA gel blot analysis of AtSPT ,SAG12and SAG13in pTRV2infected plants and pTRV2-AtCDC5infected plants.Equal loading of the gel was con?rmed by EtBr staining (bottom panel).Noting that SAG12was not up-regulated in the AtCDC5VIGS plants.
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4.2.Cloning of AtCDC5cDNA
The cDNA of AtCDC5gene was cloned with the primers (50
-GAA GAC ATG TCA GAC AGG AT-30and 50-AAC ATG GGC TAT GGC TAT GTC-30)in an intensive large-scale cloning of Arabidopsis transcription factors [9,13].
4.3.GFP fusion protein analysis
To construct GFP-AtCDC5fusion protein,the coding sequence of AtCDC5was ampli?ed by PCR using a high-?delity DNA polymerase (Pyrobest polymerase,Takara).The primers used were as follows:
AtCDC5-F:50-GCC GGA TCC ATG AGG ATT ATG ATT AAG GGA G-30
AtCDC5-R:50-GCT CTA GAT TAT GCA GAA GCT TCC ATG GCT ATG GC-30
The underlined nucleotides correspond to Bam HI and Xba I restriction sites respectively.The PCR product was then cloned into pRTL2-GFP [18],and sequencing con?rmed.
Transient transformation of onion epidermis cells and exami-nation of green ?uorescence was performed as described previously [19].
4.4.Trans-activation activity assays
Full-length and deleted coding regions of AtCDC5were ampli?ed by PCR using Pfu DNA polymerase (Takara).The primers used were listed as follows:
AtCDC5(1)-F:50-GCG AAT TCA TGA GGA TTA TGA TTA AGG G-30
AtCDC5(335)-F:50-GCG AAT TCG CAC TTT TGG CAA ATT ACT C-30
AtCDC5-R:50-GCT GTC GAC TTA TGC AGA AGC TTC CAT GGC TAT-30
The numbers in the brackets indicate the corresponding amino acid sites of respective primers,underlined nucleotides correspond to Eco RI and Sal I restriction sites,respectively.The PCR products were fused in-frame to the sequences encoding GAL4DNA-binding domain on pYF503
and
Fig.5.AtCDC5VIGS in npr1and nahG plants.(A)and (B)npr1plants,19days after Agrobacterium in?ltration.(A)npr1plants infected by pTRV2alone;(B)npr1plants infected by pTRV2-AtCDC5.(C)and (D)nahG plants,16days after Agrobacterium in?ltration.(C)nahG plants infected by pTRV2alone.(D)nahG plants infected by pTRV2-AtCDC5.
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con?rmed by sequencing.For the trans-activation activity as-says,pYF503and its derivates were transformed into yeast strain EGY48containing the reporter vector pG221[37].The positive clones containing different pYF503derivates were de-termined for their b-galactosidase activities.LiAc-mediated yeast transformations and b-galactosidase?lter assays were performed according to Clontech Yeast Protocols Handbook (Palo Alto,CA).
4.5.Construction of pTRV2-AtCDC5
pTRV2used for virus induced gene silencing has been re-ported[20]and kindly provided by Dr.Dinesh-Kumar of Yale University.For speci?cally silencing AtCDC5,a365-bp frag-ment(637e1001)of the AtCDC5cDNA was ampli?ed by PCR with the primers containing Eco RI and Xho I restriction sites indicated by underlines:F:50-CGG GAA TTC CTT TTG AAA AG-30and R:50-CGG CTC GAG TAG CAG CAC TGC-30.The resulting PCR product was cloned into pTRV2to generate pTRV2-AtCDC5.
4.6.Agro-in?ltration
The plasmid of pTRV1,pTRV2and pTRV2-AtCDC5were separately introduced into Agrobacterium strain GV3101. The Agrobacterium in?ltration was conducted as previously described[20].
4.7.Trypan blue staining
The trypan blue staining was performed as previously de-scribed[4,24,28].
4.8.Northern blot and RT-PCR
A fragment was ampli?ed by PCR from AtCDC5,AtSPT, SAG13and SAG12,with the following primers,and the result-ing PCR products were used as probes in northern blots: AtCDC5:50-AAG GCG GAG GAA GAA GCA-30
50-AACATGGCTATGGCTATGTC-30
AtSPT:50-GAT CTG CTT GGC GCA TGA GG-30
50-TGA GTG AGA TGC GGA TAT GC-30
SAG13:50-TTG GAG GCA TGA CCG CTC TT-30
50-AAC AGT GGC ACC GCC GTC AA-30
SAG12F:50-TCT CGT CCA CTC GAC AAT GA-30
50-ATG GCA AGA CCA CAT AGT CC-30
Primers for RAN1(AT5G44790)used in RT-PCR were as follows:
RAN1:50-AAC GAA GCG AAC GCC GAT GAT A-30
50-CAA GCT GCA CAA GTC ATA CCT C-30
RNA extraction,northern blot and RT-PCR analysis were carried out as previously reported[29,30].Acknowledgments
The authors thank Dr.Dinesh-Kumar(MCDB Department, Yale University)for kindly providing the VIGS vectors,and Dr.Shuhua Yang for npr1and nahG seeds.This research was supported by NSFC grants(GN30470358and 30625002)to L.-J.Qu.
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