Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance

In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways in Arabidopsis thaliana were selected for VIGS experiments in wheat. These include Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8’-hydroxylase), and Sal1 (inositol polyphosphate 1-phosphatase). Gene homologues for these three genes were identified in wheat and cloned in the viral vector barley stripe mosaic virus (BSMV) in the antisense direction, followed by rub inoculation of BSMV viral RNA transcripts onto wheat plants. Quantitative real-time PCR showed that VIGS-treated wheat plants had significant reductions in target gene transcripts. When VIGS-treated plants generated for Era1 and Sal1 were subjected to limiting water conditions, they showed increased relative water content, improved water use efficiency, reduced gas exchange, and better vigour compared to water-stressed control plants inoculated with RNA from the empty viral vector (BSMV0). In comparison, the Cyp707a-silenced plants showed no improvement over BSMV0-inoculated plants under limited water condition. These results indicate that Era1 and Sal1 play important roles in conferring drought tolerance in wheat. Other traits affected by Era1 silencing were also studied. Delayed seed germination in Era1-silenced plants suggests this gene may be a useful target for developing resistance to pre-harvest sprouting.

[1]  Angela Sample,et al.  Molecular tailoring of farnesylation for plant drought tolerance and yield protection. , 2005, The Plant journal : for cell and molecular biology.

[2]  C. Cakir,et al.  EVALUATING THE ABILITY OF THE BARLEY STRIPE MOSAIC VIRUS-INDUCED GENE SILENCING SYSTEM TO SIMULTANEOUSLY SILENCE TWO WHEAT GENES , 2008 .

[3]  J. Fry,et al.  Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection , 2003, Plant Cell Reports.

[4]  P. Langridge,et al.  Genetic and genomic tools to improve drought tolerance in wheat. , 2010, Journal of experimental botany.

[5]  I. Petty,et al.  Infectious barley stripe mosaic virus RNA transcribed in vitro from full-length genomic cDNA clones. , 1989, Virology.

[6]  M. Bruun-Rasmussen,et al.  Stability of Barley stripe mosaic virus-induced gene silencing in barley. , 2007, Molecular plant-microbe interactions : MPMI.

[7]  R. Serraj,et al.  Drought-resistant rice for increased rainfed production and poverty alleviation: a concept note. , 2009 .

[8]  Tao Chen,et al.  Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection. , 2007, The Plant journal : for cell and molecular biology.

[9]  J. Dubcovsky,et al.  A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat , 2009, BMC Plant Biology.

[10]  G. Farquhar,et al.  Isotopic Composition of Plant Carbon Correlates With Water-Use Efficiency of Wheat Genotypes , 1984 .

[11]  M. Deyholos,et al.  Transcriptional profiling of hexaploid wheat (Triticum aestivum L.) roots identifies novel, dehydration-responsive genes. , 2007, Plant, cell & environment.

[12]  B. Pogson,et al.  Identifying photoprotection mutants in Arabidopsis thaliana. , 2004, Methods in molecular biology.

[13]  Luigi Cattivelli,et al.  Transcriptional profiling in response to terminal drought stress reveals differential responses along the wheat genome , 2009, BMC Genomics.

[14]  T. Sharkey,et al.  Stomatal conductance and photosynthesis , 1982 .

[15]  Piero Carninci,et al.  Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray , 2001, Plant Cell.

[16]  C. Pikaard,et al.  Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. , 2003, The Plant journal : for cell and molecular biology.

[17]  S. Holzberg,et al.  Barley stripe mosaic virus-induced gene silencing in a monocot plant. , 2002, The Plant journal : for cell and molecular biology.

[18]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[19]  S. Datta,et al.  Effect of Water Deficit Stress on Diffusive Resistance, Transpiration, and Spikelet Desiccation of Rice (Oryza sativa L.) , 1993 .

[20]  Y. Kamiya,et al.  The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′‐hydroxylases: key enzymes in ABA catabolism , 2004, The EMBO journal.

[21]  Z. Pei,et al.  Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss. , 1998, Science.

[22]  K. Shinozaki,et al.  CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana. , 2006, The Plant journal : for cell and molecular biology.

[23]  Peter Kareiva,et al.  Genome Plasticity a Key Factor in the Success of Polyploid Wheat Under Domestication , 2007 .

[24]  J. Leach,et al.  Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance. , 2010, Plant biotechnology journal.

[25]  Yule Liu,et al.  Efficient Virus-Induced Gene Silencing in Arabidopsis1 , 2006, Plant Physiology.

[26]  B. Gill,et al.  Development of a Virus-Induced Gene-Silencing System for Hexaploid Wheat and Its Use in Functional Analysis of the Lr21-Mediated Leaf Rust Resistance Pathway1 , 2005, Plant Physiology.

[27]  V. Knauf,et al.  TILLING moves beyond functional genomics into crop improvement , 2005, Transgenic Research.

[28]  D. Ohta,et al.  Arabidopsis CYP707As Encode (+)-Abscisic Acid 8′-Hydroxylase, a Key Enzyme in the Oxidative Catabolism of Abscisic Acid1 , 2004, Plant Physiology.

[29]  Travis W. Banks,et al.  Leaf rust resistance gene Lr1, isolated from bread wheat (Triticum aestivum L.) is a member of the large psr567 gene family , 2007, Plant Molecular Biology.

[30]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[31]  M. Shah,et al.  Variation in genotypic responses and biochemical analysis of callus induction in cultivated wheat. , 2009, Genetics and molecular research : GMR.

[32]  S. Pang,et al.  Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens , 1997, Plant physiology.

[33]  J. Layton,et al.  Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat , 2003, In Vitro Cellular & Developmental Biology - Plant.

[34]  Erik Willems,et al.  Standardization of real-time PCR gene expression data from independent biological replicates. , 2008, Analytical biochemistry.

[35]  David C Baulcombe,et al.  Virus-induced gene silencing in plants. , 2003, Methods.

[36]  J. Schroeder,et al.  Hypersensitivity of Abscisic Acid–Induced Cytosolic Calcium Increases in the Arabidopsis Farnesyltransferase Mutant era1-2 Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010448. , 2002, The Plant Cell Online.

[37]  Ernst-Detlef Schulze,et al.  Carbon Dioxide and Water Vapor Exchange in Response to Drought in the Atmosphere and in the Soil , 1986 .

[38]  P. McCourt,et al.  A Protein Farnesyl Transferase Involved in Abscisic Acid Signal Transduction in Arabidopsis , 1996, Science.

[39]  Adam J. Carroll,et al.  The nucleotidase/phosphatase SAL1 is a negative regulator of drought tolerance in Arabidopsis. , 2009, The Plant journal : for cell and molecular biology.

[40]  A. Rodríguez-Navarro,et al.  The SAL1 gene of Arabidopsis, encoding an enzyme with 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast. , 1996, The Plant cell.

[41]  P. McCourt,et al.  A Novel Role for Protein Farnesylation in Plant Innate Immunity1[C][W][OA] , 2008, Plant Physiology.

[42]  Rebecca Griffiths,et al.  Shoot-specific down-regulation of protein farnesyltransferase (alpha-subunit) for yield protection against drought in canola. , 2009, Molecular plant.

[43]  Gustavo A. Slafer,et al.  Breeding for Yield Potential and Stress Adaptation in Cereals , 2008 .

[44]  Chengcai Chu,et al.  Abscisic acid and the pre-harvest sprouting in cereals , 2008, Plant signaling & behavior.

[45]  X. Huang,et al.  CAP3: A DNA sequence assembly program. , 1999, Genome research.