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Expression of a truncated form of yeast ribosomal protein L3 in transgenicwheat improves resistance to Fusarium head blight
Rong Di
a,
*, Ann Blechl
b
, Ruth Dill-Macky
c
, Andrew Tortora
d
, Nilgun E. Tumer
a
a
Biotechnology Center for Agriculture and the Environment, The School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ 08901-8520, USA
b
Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, Albany, CA 94710-1105, USA
c
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108-6030, USA
d
Undergraduate Biology Program, Rutgers University, New Brunswick, NJ 08901-8520, USA
1. Introduction
Fusariumheadblight(FHB)causedbyseveral
Fusarium
species,primarily
Fusarium graminearum
Schwabe in the U.S., is aneconomically important disease of wheat and barley worldwide[1]. It was estimated that several severe FHB outbreaks in U.S.wheatandbarleyfrom1991to1997resultedin$4.8billionoftotaldirect losses[2]. The contamination of wheat, barley and maizerelated products with the trichothecene mycotoxin deoxynivale-nol (DON) due to the infection with
F. graminearum
poses a healththreat to humans and animals[3,4]. Disruption of the
F. graminearum
gene
Tri5
, which encodes the trichodiene synthasethat catalyzes the first step in the DON biosynthetic pathway,reduces FHB severity on greenhouse- and field-grown wheat[5,6].The
Tri5
revertant strains of 
F. graminearum
regain their aggres-siveness on field-grown wheat[6]. These results have providedevidence that DON acts as a virulence factor in FHB.Trichothecene mycotoxins interact with the peptidyltransfer-ase center of eukaryotic ribosomes and inhibit protein synthesis[7].Trichotheceneshave beenreportedto havediverseroles inthecell that are not limited to the inhibition of protein synthesis. Arecent genome-wide screen in
Saccharomyces cerevisiae
revealed acritical role for the mitochondria in the toxicity of a trichothecenemycotoxin[8]. The
tcm1
mutation that conferred resistance to thetrichothecene, trichodermin was identified in the yeast
RPL3
gene,whichencodestheribosomalproteinL3[7,9,10].L3participatesintheformationofthepeptidyltransferasecenter[11,12].Therehavebeen attempts to over-express the
RPL3
gene carrying the
tcm1
mutation (W255C) in transgenic plants to achieve resistance toDON. A modified rice
RPL3
cDNA containing the W255C mutationwas transformed into tobacco[13]. The transgenic tobacco calliand protoplasts displayed greater regeneration efficiency andviability in the presence of DON. The expression of a tomato
RPL3
cDNA with the
tcm1
mutation in transgenic tobacco plantsimproved the ability of these plants to adapt to DON, but didnot result in constitutive resistance, possibly because the mutantprotein did not accumulate in the transgenic plants[14].We cloned the two different L3 genes (
RPL3A
and
RPL3B
) fromtobaccoandshowedthattheirexpressioniscoordinatelyregulated[15]. Increasing L3 levels in transgenic tobacco resulted in leaf overgrowthandmottling,andledtoanincreaseincellnumberand
Plant Science 178 (2010) 374–380
A R T I C L E I N F O
 Article history:
Received 7 December 2009
Received in revised form 2 February 2010
Accepted 3 February 2010
Available online 11 February 2010
Keywords:
Fusarium head blightWheat and barley scabTrichothecene mycotoxinDeoxynivalenolTransgenic wheat plantsRibosomal protein L3
A B S T R A C T
Fusarium head blight (FHB) is a disease that causes major economic losses in wheat and barleyproduction worldwide. Contamination of food with the trichothecene mycotoxin deoxynivalenol (DON)produced by
Fusarium
is a major health concern for humans and animals because trichothecenes arepotent cytotoxins of eukaryotic cells. Trichothecene mycotoxins inhibit translation by targetingribosomal protein L3 at the peptidyltransferase center. We previously showed that expression of an N-terminal fragment of yeast L3 (L3
D
) in transgenic tobacco plants reduced the toxicity of DON. Here, weproduced transgenic wheat plants that express the same yeast L3 (L3
D
) fragment and evaluated theirsusceptibility to
Fusarium graminearum
infection and their ability to accumulate DON. Following
F. graminearum
infection in greenhouse tests, two transgenic wheat lines expressing the highest levels of L3
D
showedreductionsindiseaseseverityandkernelDONlevels,compared tonon-transformedplants.In a field test, a transgenic wheat line with the highest L3
D
expression controlled by the maize
Ubi1
promoter had significant reductions in visually scabby kernels and kernel DON levels. These resultsdemonstrate that expression of a modified form of the ribosomal protein that is the target of DON canimprove FHB resistance in wheat.
ß
2010 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Biotechnology Center for Agriculture and theEnvironment, The School of Environmental and Biological Sciences, RutgersUniversity, 59 Dudley Rd., Rm 127 Foran Hall, New Brunswick, NJ 08901-8520,USA. Tel.: +1 732 932 8165x183; fax: +1 732 932 6535.
E-mail address:
di@aesop.rutgers.edu(R. Di).
Contents lists available atScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
0168-9452/$ – see front matter
ß
2010 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.plantsci.2010.02.003
 
a decrease in cell size. The rRNA precursor and the mature rRNAsaccumulated in these plants, suggesting that ribosome biogenesiswas up-regulated. In contrast, L3 deficiency led to a reduction incell number and an increase in cell size[15]. Since alteringendogenous L3 expression led to an abnormal phenotype intobacco, we expressed the full length and a truncated form of theyeast L3 gene corresponding to the first 99 amino acids of L3 intobacco to determine if expression of the yeast L3 gene confersresistancetoDON.TransgenictobaccoplantsexpressingyeastL3
D
were phenotypically normal and, in a germination test, showedresistance to DON and to pokeweed antiviral protein (PAP), whichalso targets L3[16]. PAP is a 29-kDa ribosome inactivating protein(RIP) isolated from pokeweed plants that inhibits translation bybinding to L3 and catalytically removing a specific purine residuefrom the highly conserved
a
-sarcin/ricin loop (SRL) of the largerRNA[17]. Ribosomes from tobacco plants expressing PAP andyeast L3
D
were not depurinated, even though PAP was associatedwith ribosomes. These results demonstrated that yeast L3
D
conferred resistance possibly by protecting ribosomes from thetranslation inhibitory effects of DON and PAP[16].In this study, we aimed to test whether expression of the yeastL3
D
gene could protect wheat ribosomes from DON and thusreduce FHB severity in wheat plants carrying L3
D
transgenes.Bread wheat (
Triticum aestivum
=
Ta
) is hexaploid and thuscontains six different
RPL3
genes:
TaRPL3A1
,
TaRPL3A2
,
TaRPL3A3
,
TaRPL3B1
,
TaRPL3B2
and
TaRPL3B3
[18].Wetransformedhexaploidwheat cultivar Bobwhite with yeast L3
D
under the control of twodifferent promoters and assayed the resultant transformants for
RPL3
and L3
D
expression levels and for FHB resistance and DONaccumulation in the greenhouse and field.
2. Materials and methods
 2.1. Production of transgenic wheat plants
The coding sequence of the N-terminal 99 amino acids of yeastribosomalproteinL3wasclonedinplaceofthe
bar 
codingregioninthe wheat expression vector pUBK[19]. The resulting construct,called pLem-L3d, contained the L3
D
gene between the barleytissue-specific
Lem1
promoter[20]and the nopaline synthase(NOS) 3
0
transcription terminator (Fig. 1). The L3
D
gene was alsoclonedintoasimilarwheatexpressionvectorcontainingthemaize
Ubi1
promoter/first intron[21], resulting in construct pUbi-L3d(Fig. 1).Embryogenic calli of a hexaploid FHB-susceptible spring wheat(
T. aestivum
L. em. Thell. cv Bobwhite) were co-bombarded with a2:1(pLem-L3d)or4:1(pUbi-L3d)molarratiooftheL3
D
constructsand pUBK containing the
bar 
selectable marker gene under thecontrol of 
Ubi1
. Regenerants were selected with bialaphos asdescribedpreviously[19,22],exceptthatthecallusrecoverymediaafter bombardment contained 5
m
M CuSO
4
and the regenerationmedia contained 5
m
M CuSO
4
, 0.1 mg l
À
1
6-benzylamino purine,and3 mg l
À
1
bialaphos.T
0
plantscontainingpLem-L3dorpUbi-L3dwere identified using PCR of wheat genomic DNA, as describedpreviously[19], with forward primer UbiA2[19]or Lem752 [5
0
-GACAGTGGGAGTGGGGTTTG-3
0
] in combination with reverseprimer L3-L3d [5
0
-CGACGTAACCGACAACACC-3
0
] and an annealingtemperature of 62
8
C.
 2.2. Real-time PCR analysis to determine the gene expression levels intransgenic wheat plants
Total RNA was isolated from the florets and immature seeds orleaves of transgenic wheat plants using TRIzol
1
Reagent (Invitro-gen) according to the manufacturer’s protocol. The RNA sampleswere treated with RQ1 RNase-Free DNase (Promega) following themanufacturer’s instructions and further purified with phenol/chloroform extraction and ethanol precipitation. SuperScript
1
reversetranscriptase(Invitrogen)andoligodTwereusedtoproducethe first strand cDNA from 5
m
g of total RNA. ABI PRISM 7000Sequence Detection System (Applied Biosystems) was used toperform real-time PCR analysis using gene-specific primersdesigned by ABI Primer Express software, following the manufac-turer’s protocols. The primers for yeast L3
D
were L3d67F, 5
0
-GCCTCCATCAGAGCTAGAGTTAAGG-3
0
and L3d145R, 5
0
-AACCCAA-GAAGGAAGTTAGAGCAA-3
0
.Theprimersusedtomeasurethelevelsof wheat
TaRPL3A
were WL3A829F, 5
0
-CACCGCACAGAGATGAA-CAAA-3
0
and WL3A900R, 5
0
-AGTGCAGGCCTCGTGAGACTC-3
0
. Theprimers for the wheat
TaRPL3B
were WL3B829F, 5
0
-CACCGAACT-GAGATGAACAAG-3
0
and WL3B900R, 5
0
-GGTAGAGGCATCAT-GAGTTTC-3
0
. The relative gene expression levels of 
TaRPL3
weremeasured using wheat
a
-tubulin (GenBank Access #U76558) as aninternal control with primers WTub942F, 5
0
-CAGCTGAGAAGGCT-TACCATGA-3
0
and WTub1000R, 5
0
-AAAGGCGCTGTTGGTGATCT-3
0
.The 2
À
DD
C 
T
method[23]was used to determine the relative geneexpression levels.
 2.3. GreenhousescreeningoftransgenicwheatlinesforresistancetoF. graminearum
Plants were grown in pots (15 cm
Â
15 cm
Â
16.5 cm; BeldenPlastics, Roseville, MN) containing commercial potting medium(Metromix200,ScottsCo.)inagreenhousemaintainedat20
Æ
2
8
Cwith a 16-h light and 8-h dark cycle. Six to seven seeds were plantedin each pot. Five pots per line were thinned to six plants at Zadoksgrowth stage (GS) 13–14. In addition to the transgenic lines, twocheck lines were included in the greenhouse screening. The FHBchecks used were Wheaton, a hard red spring wheat, releasedcooperatively by the Minnesota Agricultural Experiment Station andUSDA-ARS in 1984, which is highly susceptible to FHB, and Alsen, ahard red spring wheat released from North Dakota State Universityand moderately resistant to FHB. The FHB resistance in Alsen isderived from the Chinese wheat Sumai 3. Bobwhite was included asan untransformed control. The planting was replicated. Each pot wasfertilized with 5.8 g of slow release 14–14–14 (N–P–K) fertilizer(Osmocote Classic, Scotts Co.) at GS 13–14. Insect pests werecontrolled with an application of imidacloprid (Marathon 60WP,Olympic Hort. Products). Powdery mildew was controlled with asingle application of tridimefon (Bayleton 50% DF, BayerCropScience).
Plants were inoculated when the main spike from each plantreached anthesis (GS 60–65). A single spikelet at the central nodeof the main spike of each plant was inoculated with 10
m
l of amacroconidial spore suspension (100,000 conidia ml
À
1
) of 
F. graminearum
using a repeating 500-
m
l Hamilton 700 syringe(model 80830), fitted with a PB600 dispenser (model 83700)(Hamilton Co.). Twelve isolates of 
F. graminearum
collected fromcommercial fields of wheat and barley in 2002 and 2003, whichwerenaturallyinfectedwith
F.graminearum
,wereusedtoproducethe inoculum according to the previously described procedure
Fig. 1.
Constructs used to generate the transgenic wheat plants. A gene fragmentfrom yeast that encodes the N-terminal 99 amino acids (L3
D
) of the ribosomalproteinL3wasclonedintowheatexpressionvectorsunderthecontrolofeitherthebarley
Lem1
(pLem-L3d) or the maize
Ubi1
promoter/first intron (pUbi-L3d). Thenopaline synthase (NOS) 3
0
transcription termination sequence was used as thetranscription terminator in both constructs.
R. Di et al./Plant Science 178 (2010) 374–380
375
 
[24]. Immediately after inoculation, the plants were placed in adew chamber for 72 h. Following the dew period, the plants werereturned to the greenhouse bench. Disease development wasassessed visually 21 days after the inoculation. FHB incidence isdefined as the percentage of spikes with visually symptomaticspikelets and FHB severity as the percentage of symptomaticspikelets from the total spikelets observed. The DON content of grain was determined on the kernels harvested from the fourspikelets adjacent to the inoculated spikelet of inoculated heads.Heads were harvested at maturity and threshed by hand.
 2.4. Field testing of transgenic wheat lines for resistance to F. graminearum
The 2008 field screening nursery was located at UMore Park,Rosemount, MN. In addition to the transgenic wheat lines,untransformed Bobwhite, the moderately resistant wheat Alsenand the FHB-susceptible wheat Wheaton were included as checks.The experimental design was a randomized block with fourreplicates. Plots were 2.4 m long single rows. All plots, except anon-inoculated Wheaton check, were inoculated twice. The firstinoculation was applied at anthesis and the second 3 days afteranthesis. Inoculum was a composite of 41
F. graminearum
isolatesat a concentration of 100,000 macroconidia ml
À
1
. Polysorbate 20(Tween 20) was added at 2.5 ml l
À
1
to the inoculum as a wettingagent. The inoculum was applied using a CO
2
-powered backpacksprayer fitted with a SS8003 TeeJet spray nozzle with an output of 10 ml s
À
1
at a working pressure of 275 kPa. FHB incidence andseverity were assessed visually 20–25days after inoculationon 20arbitrarily selected spikes per plot. FHB incidence and severitywere defined as stated above. The harvested seeds from each plotwere split using a Borrner divider (Seedburo) to obtain a 50 g sub-sample,whichwasthencleanedbyhand.Thesesampleswereusedto estimate the percentage of visually scabby kernels (VSK%),according to method of Jones and Mirocha[25], and then analyzedfor deoxynivalenol (DON).
 2.5. Determination of DON level in kernels of the inoculated wheat  plants
Grain samples were analyzed for DON according to thepublished protocol[26]. Briefly, the samples were ground for2 minwithaSteinLaboratoriesMill(modelM-2,SteinLaboratoriesInc.).DONwasextractedfrom4 gofthegroundsamplein16 mlof acetonitrile–water (84:16, vol/vol) placed on an Eberbach recipro-cal shaker (model 6010, Eberbach) for 1 h. A 1-ml sample elutedthrough a specially prepared cleanup column[27]was evaporatedtodrynesswithnitrogen.DONwasderivatizedandanalyzedusinggas chromatography–mass spectrometry (GC/MS) (Model QP-2010,ShimadzuLtd.)inselectedionmonitoring(CSIM)mode.DONconcentration was determined based on retention times and peakareas by comparing to standards.
3. Results
 3.1. Generationoftransgenicwheatplantsexpressingyeastribosomal protein L3
D
To determine if expression of L3
D
in transgenic wheat wouldconfer resistance to DON, embryogenic calli of susceptible springwheat(
T. aestivum
L.em.Thell.cvBobwhite)weretransformed
via
particle gun bombardment with genes encoding L3
D
under thecontrolofeitherthebarley
Lem1
[20](pLem-L3d)orthemaize
Ubi1
[21]promoter (pUbi-L3d) (Fig. 1). The
bar 
gene, conferringresistance to the herbicides bialaphos and BASTA, under controlof 
Ubi1
,wasco-bombardedwiththeL3
D
constructsintothecalliasa selection marker to identify the stably transformed T
0
plants.SeveralbialaphosresistantT
0
wheatplantswereregeneratedfromone bombardment experiment with each expression vector. Someof these T
0
plants also contained
Lem1
::L3
D
or
Ubi1
::L3
D
, asdetermined by PCR with oligonucleotide primers specific for thepromoters and L3
D
(data not shown). The integration of the L3
D
transgenes was confirmed by PCR analyses of genomic DNA fromT
1
plants, demonstrating that the transgene locus was inherited(data not shown). All (12/12) the progeny of one T
0
plantcontaining
Lem1
::L3
D
inherited a
Lem1
::L3
D
transgene, suggest-ing that the original transformant contained at least twoindependent transgenic loci. Three homozygous T
2
sublinesderived from this event were identified by PCR (data not shown)and named 771, 772 and 774. The progeny of two independent T
0
plants containing
Ubi1
::L3
D
segregated 3:1 (8/10 and 11/15) forthe transgene, suggesting they each contained a single transgenelocus. Homozygous T
2
plants from these two transformants wereidentified by PCR and were named 8133 and 8153. Eight to tenhomozygousT
3
plantsfromeachofthefivelinesweregrowninthegreenhouse for expression and resistance analyses. No develop-mental or seed set differences were observed between thetransgenic plants and their parent.
 3.2. Expression of yeast L3
D
in transgenic wheat plants
For each of the five homozygous transgenic wheat lines,expression of the L3
D
transgene was measured at anthesis, thestage at which they would be most susceptible to
Fusarium
infection.Thefloretsandimmatureseedsweresampledsincemostof the natural infection occurs in these organs. Total RNA wasextracted from these samples and used in real-time polymerasechain reaction (PCR) assays to measure the expression level of theyeast L3
D
gene, using a set of primers specifically designed for the5
0
end of the yeast
RPL3
gene, corresponding to the N-terminalaminoacids+22to+48ofL3.AsshowninFig.2A,theseaminoacidsare significantly divergent among the yeast and TaRPL3 proteins.Experimental results showed that these primers did not hybridizeto the endogenous wheat
TaRPL3
genes (data not shown). TherelativeL3
D
expressionlevelsweredeterminedusingthewheat
a
-tubulin mRNA as an internal control. Real-time PCR analysis wasperformed on RNA from four to five different plants of eachhomozygous line with two replicates per plant and results wereaveraged. As shown inFig. 3A, the L3
D
expression was detected athigh levels in the florets/immature seeds of transgenic wheatplants. Transgenic lines 772 and 8153 contained the highest levelsof the yeast L3
D
transcripts.
 3.3. Relative steady state levels of the endogenous wheat TaRPL3transcripts in transgenic wheat plants
We have shown previously that the endogenous
TaRPL3
geneswere up-regulated in the transgenic tobacco plants expressing theyeastL3
D
[16].InordertodetermineifexpressionoftheyeastL3
D
affected expression of the endogenous
TaRPL3
genes in wheat, wemeasured the
TaRPL3
expression levels relative to those of 
a
-tubulin in the homozygous lines of transgenic wheat plants. The
TaRPL3
gene family consists of three alleles of both
TaRPL3A
and
TaRPL3B
[18]. The following six different wheat
TaRPL3
genesequences from the GenBank were used in this study:
TaRPL3A1
(accession #AY343327),
TaRPL3A2
(accession #AY343328),
TaR-PL3A3
(accession #BK001237),
TaRPL3B1
(accession #BK001235),
TaRPL3B2
(accession #BK001234) and
TaRPL3B3
(accession#AY347532). Comparison of the amino acid sequences amongthe three wheat
TaRPL3A
genes and three wheat
TaRPL3B
genesshowed that TaRPL3A and TaRPL3B protein sequences are verysimilar within the A and B groups. However, the sequences of the
R. Di et al./Plant Science 178 (2010) 374–380
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