Evaluation of a Brassica napus Auxin-Repressed Gene Induced by Flea Beetle Damage and Sclerotinia sclerotiorum Infection

Biotic stresses negatively affect canola growth and production. Flea beetle damage and Sclerotinia sclerotiorum (S. sclerotiorum) infection are two of the worst biotic stresses for canola. Auxin Repressed Proteins (ARPs) responsive to several abiotic stresses have been reported. However, information about ARPs induced by Flea beetle damage and S. sclerotiorum infection, their roles in biotic stress tolerance are still lacking in canola. ESTs for an Auxin Repressed Protein 1 (BnARP1) were highly represented (expressed) in a Brassica napus subtractive library developed after leaf damage by the crucifer flea beetle (Phyllotreta cruciferae). Expression of this gene was under different developmental control in B. napus, and it was co-induced in B. napus by flea beetle feeding, S. sclerotiorum infection, drought and cold. A total of 25 BnARP genes were represented in different B. napus stress and development EST libraries and indicated larger, diversified families than known earlier. Dwarf phenotypes, primary root growth inhibition, lateral root enhancement, reduced sensitivity to 2, 4-D, and reduced PIN1 and LOX expression in transgenic Arabidopsis expression lines suggest that BnARP1 is an auxin repressor that prevents auxin transport and supports an interaction between the auxin and jasmonate signalling pathways. And the increased survival after S. sclerotiorum infection in transgenic over-expression Arabidopsis suggests that BnARP1 could play a role in S. sclerotiorum tolerance through connecting auxin and jasmonate signalling pathways.


Introduction
Plant resistance or tolerance to biotic stress consists of constitutive or induced defense mechanisms, and inducible defense is thought to be more durable than constitutive defense [1] [2].From a molecular perspective, it is clear that a plant's response to challenge by insects and fungi is mediated by a network of cross-talking pathways and not by simple linear signal transduction cascades [2].This network includes the jasmonic acid (JA), salicylic acid (SA), ethylene, abscisic acid (ABA) and auxin signalling pathways and presumably enables a coordinated, specific response.JA and SA function as key signaling molecules, which activate distinct sets of defense-related genes in response to herbivore damage.
For example, aphid feeding induces SA-dependent transcription of Pathogenesis-Related protein 1 (PR-1) and β-1, 3-glucanase (BGL2), as well as Plant Defensin Factor 1.2 (PDF1.2) and Lipoxygenase 2 (LOX2), both of which are involved in the JA signaling cascade [3] [4].JA and ethylene can be either synergistic or antagonistic hormones in terms of the expression of defensive genes in response to insect attack [5] [6] [7] [8], while both of these hormones mediate against pathogen attack (partly by defense gene induction) [9].Curiously, plants deficient in ethylene signaling show either increased susceptibility or increased resistance, depending on the plant and pathogen [10].ABA-dependent signaling is also well known for regulating abiotic stress-induced gene expression [11], and ABA antagonizes JA/ethylene-responsive defense gene expression and modulates disease resistance in Arabidopsis [12].
Auxin plays a central role in the growth and development of plants, including stem elongation, lateral branching of roots and shoots, establishment of embryonic polarity, and vascular development [13].Recent studies in Arabidopsis have shed light on several gene families involved in the auxin signaling pathway [14] [15].These include the Auxin/Indole-3-Acetic Acid repressor family (Aux/ IAAs) which encodes short-lived nuclear proteins that regulate auxin responsive genes through dimerization between family members and interaction with the auxin response factor (ARF) family [16] [17].ARFs bind to auxin response elements (AREs) in the promoters of auxin-inducible genes to regulate their expression [18].Ethylene Insensitive Root 1 (EIR1) is one of the PIN-FORMED (PIN) family of auxin efflux carriers which are responsible for the active, directional transport of auxin through plant tissues [19].A defective response to jasmonate in the auxin-signaling mutant axr1 (AUXIN-RESISTANCE 1 gene) provides a mechanistic link between JA and auxin-signaling pathways and integration of diverse defense pathways mediated by JA, ethylene and SA [20].
Auxin repressed proteins (ARPs) are also known.These include developmentrelated ARPs such as the fruit repression SAR5 from strawberry [21], the dormancy-associated SbDRM1 from sorghum and pea [22] [23], a germination-related gene from tobacco [24], a hypocotyl elongation-related RpARP from black locust [25], the auxin-induced EuNOD-ARP1 gene from Elaeagnus umbellate root nodules [26], and the cucumber CsGRP1, which accumulates on the upper L. M. Wu et al. side of seedlings during gravimorphogenesis [27].Stress-responsive ARPs include the drought/heat-induced AtARP1 from Arabidopsis [28], a gene induced by salinity and cold from hot pepper [29], five genes responsive to drought/ cold/high-salinity from chickpea [30], the salinity/drought-responsive BnARP from B. napus [31], and two genes responsive to chilling, heat shock and salt stress (BrARP1 and BrDRM1) from B. rapa [32].However, the role of ARPs that mediate resistance or susceptibility to insects and fungi is unknown.
In the present study, we explored the roles of one auxin-repressed BnARP1 gene.BnARP1gene was strongly induced under several common stress conditions.Curiously, the introduction of the BnARP1 transgene inhibited apical dominance and primary root growth, and increased lateral root numbers when expressed in Arabidopsis.Expression in Arabidopsis of BnARP1 yielded plants with improved survival after infection with a fungus.

Plant Materials
Expression analysis, bioassays, and EST library development for Brassica napus were conducted using the double haploid line DH12075 (derived from a cross between the blackleg-resistant canola cultivar Cresor and the susceptible cultivar Westar, G. Seguin Schwartz and G. Rakow, formerly of AAFC Saskatoon Research Centre).Transgenic Arabidopsis over-expression (OE) lines were developed in a Columbia (Col-0) ecotype.

EST Subtraction Library Development, Screening, and
Bioinformatics Total RNA was isolated with guanidine hydrochloride from undamaged 8-weekold B. napus leaves and from flea beetle-damaged B. napus leaves of the same age.Leaves were damaged to 10-20% of the tissue mass after a 24 h laboratory feeding bioassay using wild Phyllotreta cruciferae flea beetles collected from a turnip field in Saskatoon.The two sets of RNA were treated with RQ1 RNAasefree DNAase (Promega, Madison, Wisconsin, USA) and poly (A) + RNA isolated using Oligotex (Qiagen, Toronto, Ontario, Canada) following the manufacturer's instructions.The cDNA synthesis and subtraction were performed according to the protocol provided in the PCR-select cDNA Subtraction Kit from Clontech Laboratories (Mountain View, CA, USA).In brief, the tester (damaged tissue) and driver (undamaged tissue) cDNA populations were digested separately with Rsa I to obtain shorter (~100 -1200 bp), blunt-ended molecules.
Tester cDNA was ligated to different adaptors to create two tester populations, which were hybridized separately with excess driver cDNAs to generate the templates required for PCR amplification of differentially expressed cDNAs.PCR subtractive amplicons were cloned in a pGEM-T easy vector (Promega, Madison, WI, USA).The subtraction library was annotated by BLAST analysis to the L. M. Wu et al.
Arabidopsis database (TAIR) and analyzed by bioinformatics according to Gruber et al. [33].Other B. napus stress or development EST libraries identified in Gruber et al. were developed using a modified pSPORT vector and the Super-Script Plasmid System with Gateway Technology [33].All EST libraries were deposited at http://brassicagenomics.ca.BnARP sequences detected in the flea beetle (FB)-damaged leaf subtractive library were assessed for representation and homologues in other EST libraries using reciprocal best hit (RBH) BLAST analysis [34].Alignments of translated sequences for BnARPs were performed using the BioEdit multiple sequence alignment program (http://www.mbio.ncsu.edu/bioedit/bioedit.html).

Transformation of Arabidopsis with BnARP1 Binary Vector
BnARP1 full-length coding regions (318 bp, GenBank accession number: KM821273) were amplified by PCR from cDNA of 8-week-old B. napus leaves using primers described in Supplementary Table 1.Amplicons were sequenced and those specifying BnARP1 were then cloned as XbaI-SstI fragments downstream of the CaMV 35S promoter in binary transformation vector pBI121 (Clontech, Mountain View, CA, USA).The binary plasmids were introduced into Agrobacterium tumefaciens by electroporation.For plant transformation, wild type Arabidopsis seeds were sown on a wet masked soil mixture in pots.
Pots were placed in the dark at 4˚C for 2 days and then moved to an environmentally controlled greenhouse at 22˚C with 16 h light and 8 h dark supplemented with halogen lamps.For single binary vector transformation, flowering Arabidopsis plants were transformed with the Agrobacterium using the floral dip method [35].

Molecular Analysis
T 1 transgenic Arabidopsis plants were selected by growth on half MS plate with kanamycin and detection of the transgene fragment after PCR analysis with specific primers (Supplementary Table 1) using genomic DNA isolated from rosette leaves by alkaline lysis [36].Transgenic plants and transgene insertion patterns were confirmed by Southern blot analysis using a CTAB extraction method for genomic DNA isolation [37].Southern blotting and hybridization were performed on Hybond N nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany) as previously described [38] using 10 μg DNA (per sample) digested overnight with EcoRI, Hind III or XhoI.The 318-bp BnARP1 coding sequence described above was labeled as probes with 32 P-dCTP using a random-primer labeling kit (Invitrogen, Carlsbad, CA, USA).Seed from 10 independently transformed T 1 plants selected by PCR of the transgene were used to develop T 2 homozygous plants used for bioassays.
For Northern blot analysis of transgenic or non-transgenic plants, tissue samples were collected from a range of developmental stages or stress treatments (detailed below), then frozen immediately in liquid N 2 .Total RNA was extracted from tissues as described by Sambrook et al. [39] and separated on 1% agarose L. M. Wu et al. gels (30 μg per lane for B. napus; 10 μg for Arabidopsis).Northern membranes were prepared and hybridized as described by Sambrook et al. [39].Probes were prepared from PCR products developed using specific primers detailed in Supplementary Table 1, then labeled with 32 P-dCTP as above.Membranes were washed twice at 65˚C in 2X SSC-0.5% SDS for 15 min each and then for 30 min at 65˚C in 0.2X SSC-0.5% SDS with gentle shaking, followed by exposure to Xray film for 3 h.The Northern blots were replicated twice using independent RNA preparations.

Root and Auxin Assays
Transgenic Arabidopsis seedling root growth was assayed on T 2 homozygous plants growing on Murashige and Skoog (MS) agar plates as described previously [40].Plates containing seeds were chilled for 2 days at 4˚C before being placed in a continuously illuminated incubator at 21˚C.Primary root length of 20 seedlings per genotype was measured 4, 7 and 14 days later and the entire experiment repeated 3 times.For root sensitivity to auxin, 4-day-old seedlings were transferred to vertically oriented MS agar plates containing 2, 4-D (0.0 -1.6 μM).
The number of lateral roots of 10 seedlings per each genotype were counted after an additional 3 d of growth and the entire experiment was repeated 3 times [41].

Flea Beetle Feeding Bioassays
Adult Phyllotreta cruciferae flea beetles (from a spring population) were collected and maintained in white-walled cabinets (housed in clear plastic cages) on cabbage and water for up to one week at 20˚C, 16 h photoperiod, 100 μmol m −2 •s −1 , then starved for 24 h prior to using them.To obtain FB damaged leaves (for subtraction library development and Northern blots) and cotyledons (for Northern blots), B. napus plants were grown in a soil-less mixture in 10 cm pots in a greenhouse supplemented with halogen lamps for 8 weeks (leaves).To test B. napus seedling damage by FB, one week-old seedlings were grown in small cylinders (for cotyledons).Plants and seedlings were pre-selected for uniformity and then evenly spaced in a foam-based arena placed inside a 50 × 50 × 50 cm clear plastic cage and exposed to 400 flea beetles per cage for 0 -24 h in an uniformly lit, white-walled, controlled environment chamber (20˚C, 16 h photoperiod, 100 μmol•m −2 •s −1 ; 65% relative humidity).Damage on leaves or cotyledons was scored using a rating scale from 0 (denoting no damage) to 10 (denoting the entire tissue destroyed) as described in Palaniswamy et al. [42].Thus, a rating of 1 was assigned if approximately 10% of the area of the tissue was damaged.Leaf and cotyledon tissues were collected and frozen in liquid N 2 , then stored at −80˚C.For bioassays on transgenic Arabidopsis expression lines and nontransgenic lines, eight (8) T 2 seedlings per transgenic line, plus WT and empty vector control lines, were grown in randomized rows for 12 days in the same white-walled growth cabinet in 96-well micro-titre plate arenas; then bio-assayed with 50 FBs per arena fitted with a modified top and scored after 24 h for damage as described in Hallett et al. [43].Each bioassay experiment was repeated 3 times.
L. M. Wu et al.

Cold and Freezing Bioassays
One-week-old transgenic and non-transgenic Arabidopsis seedlings grown in MS were placed in a 4˚C chamber with a 16 h photoperiod (approx.100 μmol m −2 •sec −1 ) for 24 h.Non-acclimated 7-day-old T 2 transgenic Arabidopsis seedlings and cold-acclimated transgenic plants were grown in petri dishes with MS.
Plants were placed at −2˚C in the dark in controlled temperature freezing chamber for 3 h, after which freezing of the plates was nucleated with ice chips as previously described [44].The plants were then incubated an additional 21 h at −2˚C, followed by −5˚C for 24 h.The temperature was then raised to 4˚C for 24 h and plants allowed to recover at 24˚C for a further 48 h in continuous light (approx.100 μmol•m −2 •sec −1 ).The entire experiment was repeated 3 times in a randomized design and then scored for survival.

Mechanical Wounding Assay
Seeds of B. napus were sown in potting medium and grown in greenhouse pots at 22˚C with 16 h light and 8 h dark supplemented with halogen lamps for 4 weeks.Fully expanded leaves of the plants were punctured with a sterile forceps and post-wounded plants incubated for one hour.

Drought Tolerance Bioassay
Transgenic and non-transgenic Arabidopsis plants were grown in a soil-less potting mixture (one plant per pot; 40 pots per flat) in a greenhouse at 22˚C with 16 h light and 8 h dark supplemented with halogen lamps.After 3 weeks of growth with one watering per day (until draining), water was withheld for 9 days and then all pots were re-watered daily (until draining) and plant re-growth scored 4 days later.The bioassay was laid out in a randomised design, with twenty plants per line and four biological replicates, means (± standard error) were separated using t-tests at p < 0.05.

S. sclerotiorum Infection Test
A modified S. sclerotiorum infection method was used based on a spray bioassay previously described by Pedras and Ahiahinu [45].Transgenic and control Arabidopsis seedlings were grown in a potting mixture (5 seedlings in one pot representing one line) with pots placed in a randomized design in 50 cm × 25 cm trays (4 pots per line; total of 50 pots per tray).S. sclerotiorum was obtained from Dr. Fengqun Yu at the Saskatoon Research Centre, and liquid cultures were initiated with five sclerotia per 100 ml of potato dextrose broth (PDB) media and shaken (110 rpm) at 20˚C for 7 days.Trays of three-week-old Arabidopsis plants were uniformly sprayed for 1 min with the fresh S. sclerotiorum inoculum (500 ml per tray) using a hand sprayer.The pots were then incubated in a growth chamber at 24˚C with 16 h light and 8 h dark supplemented with halogen lamps for 7 days and examined visually for disease symptoms and survival.

Statistical Analysis
Analysis of variance was conducted using LSD tests in SAS ver 9.0 [46].Means (±standard error) were separated using t-tests at p < 0.05.L. M. Wu et al.

BnARPs Represent Differently in Tissue-Specific or Stress-Responsive EST Libraries
A survey was conducted of genes induced in a FB damaged leaf subtractive EST library developed from eight-week-old B. napus leaves damaged by crucifer flea beetle feeding.ESTs coding for an Auxin Repressed Protein BnARP1 were much more strongly represented in this library than in a wide range of other B. napus tissue-specific or stress-responsive EST libraries (Table 1).In fact, BnARP1 was represented by 17 ESTs (1.32%) of a total of 1292 ESTs in the FB leaf damaged library.BnARPs from the FB damaged leaf subtraction library were present in low abundance in the etiolated seedling library (0.02%), a flea beetle damaged cotyledon library (0.03%), a S. sclerotiorum infected stem library (0.09%), an early anther library (0.03%), and moderately expressed in a senescent leaf library (0.28%).We predicted that the BnARP1 is the major representative of BnARPs in the FB damaged leaf subtraction library and may function mainly in flea beetle-host plant interactions.

ARP Comprises Gene Families in Brassica napus
The B. napus flea beetle damaged leaf subtractive library included a total of 31 ARP ESTs.These ESTs were classified into distinct genes using the criteria that ESTs represented the same gene if sequences were ≥90% identical (out of a total of 200 bp) [47].The FB leaf subtractive library contained four ARP proteins   L. M. Wu et al.

BnARPs Are Differentially Regulated during Development and Stress
The flea beetle damaged subtractive leaf library was most enriched in BnARP1 (17 ESTs) and BnARP2 (11 ESTs) compared with other members of the ARP families.Hence, we were curious to find out how the most particular BnARP1 gene responded to other forms of stress.Northern blot analysis showed that transcripts detected by the BnARP1 probe were present already in undamaged mature B. napus rosette leaves (Figure 2), although they had not been represented in the young leaf EST library (Table 1).Transcripts detected by the BnARP1 probe were also strongly induced in leaves damaged by flea beetle feeding, S. sclerotiorum infection, dehydration, and by 7 h of cold shock, although BnARP6 was the only ARP EST recovered in a cold acclimated (dark) library and BnARP2 was the only member found in the S. sclerotiorum-infected stem library (1 EST).Transcripts detected by the BnARP1 probe were completely repressed by mechanical wounding (Figure 2); and it did not appear in the flea beetle damaged cotyledon library, instead, BnARP4, 7 and 8 appeared in the damaged cotyledon library (Table 1).BnARP3 was exclusively expressed in the flea beetle-damaged leaf library in addition to BnARP1, BnARP4 and BnARP2.
Since cotyledons are critical tissues impacted by flea beetle damage to the canola crop [48], we also conducted additional laboratory bioassays and Northern blots  on cotyledons fed upon by flea beetles.Here, the BnARP1 gene was only transiently induced at 8 h and 16 h after flea beetle feeding on cotyledons, and expression was no longer detectable by 24 h of feeding (Figure 3).
To confirm whether BnARP1 was also subject to tissue or development constraints, Northern blots were conducted on a range of tissues.BnARP1 was moderately detected in 8-week-old fully expanded leaves and weakly detected in mature (8-week) vegetative stem and petioles, open flowers, and seed pods, barely detected in seedling leaves, stem, or roots, and not at all detected in undamaged cotyledons (Figure 4).Curiously, undamaged young leaf and undamaged cotyledons were the only tissues without any ARP genes.This was consistent with representation in the development EST libraries, although BnARP1 ESTs did appear at very low frequencies (1 & 3 ESTs) in early anther and senescent leaf EST libraries (Table 1).

Expression of BnARP1 in Transgenic Arabidopsis and Impact on Flea Beetle Feeding, Dehydration, and Cold
Since BnARP1 was most highly induced by flea beetle-feeding and moderately induced by drought and cold temperatures, we tested whether this gene can impact plant responses to these three types of stress when expressed in transgenic Arabidopsis.Therefore, 99 BnARP1 over-expression lines (BnARP1-OE) were developed by transfection of Arabidopsis with A. tumefaciens binary vector.shown as a gel loading control.1) 7-day cotyledon, 2) 14-day seedling leaf, 3) 14-day seedling stem, 4) 14-day seedling root, 5) 8-week fully-expanded adult leaf, 6) 8-week adult stem, 7) fully-opened flower, 8) seed pod at 20 days-after-pollination, 9) 8-week adult petiole.The probe was hybridized with membrane overnight and membrane was exposed to film for 3 h.These plants showed diverse growth phenotypes and were confirmed by PCR using primers that amplified the transgene fragment (a small number of representative lines shown in Figure S2(A); Supplementary Table 3).A second band (a 519 bp AT2G33830 genomic sequence from ATG to TGA) was occasionally amplified because of similar sequences occurring between BnARP1 and AT2G33830 when using BnARP1F and BnARP1R primers (Figure S2(A), S3).We manipulated this model plant rather than B. napus, since Arabidopsis is responsive to the crucifer flea beetle [43], genetically close to B. napus [49], and grows more quickly than the crop Brassicas.Northern blots showed that the expression of transgene in 10 selected single transgene BnARP1-OE expression lines varied strongly (Figure 5).However, FB feeding, drought, and freezing bioassays on these lines showed no statistical significant differences compared with WT control Arabidopsis or transgenic control Arabidopsis expressing an empty vector binary vector (data not shown).

Phenotypes of BnARP1-OE Lines
Nearly half of the independently generated T 1 BnARP1-OE Arabidopsis seedlings (49/99) showed obvious inhibition of primary root and shoot elongation and stimulation of lateral root formation while growing on kanamycin-selective MS medium (Supplementary Table 3).Out of 10 representative T 2 homozygous lines selected for further analysis, eight lines continued this absence of root apical dominance and dwarf vegetation phenotypes after being transferred into kanamycin-free soil-less mixture for at least 7 days (Figure 6(a)-(c)), while the remaining two BnARP1-OE lines (41 and 52) showed earlier flowering than WT.
This dwarf vegetation phenotype began to recover by 14 days in the potting mixture without kanamycin, such that the eight transgenic lines appeared to be growing at a WT growth rate after 3 weeks.However, they were delayed in bolting and their seeds ripened from one-to-four weeks later than WT because of the initial growth inhibition.Leaves were taken from 14-day-old plants.Ethidium bromide stained rRNA shown as a gel loading control, and the probe was hybridized with membranes for overnight and membrane was exposed to film for 3 h.

BnARP1-OE Plants Reduced Hypocotyl Elongation
Lee et al. [32] reported that BrARP1 and BrDRM1-overexpression Arabidopsis plants showed reduced hypocotyl elongation, therefore, similar experiment was conducted to show whether BnARP1 has overlap function as that of BrARP1, which has 97.22% similarity in amino acids with BnARP1.Hypocotyl elongation was reduced in BnARP1-OE lines with and without the presence of 1 μM NAA when grown in the dark (Figure 7).These results imply that BnARP1 and BrARP1 have function overlap in hypocotyl elongation.

Auxin Affects Root Growth and Expression of the BnARP1 Transgene
Because auxin plays a role during primary root and lateral root development,  The auxin-repressed transgene RpARP from black locust (Robinia pseudoacacia) is post-transcriptionally regulated (repressed) in response to exogenous auxin applied to transgenic expression plants [25].Hence, Northern blot analysis was performed to see if the BnARP1 gene was also affected by exogenous auxin.Surprisingly, transcripts for BnARP1 accumulated strongly in BnARP1-OE-1 transgenic Arabidopsis lines after 2, 4-D application and increased even more by 2h and 6 h (Figure 8).A small part of this transcription could represent the Arabidopsis AT2G33830 gene, which has 92.59% amino acid identity with BnARP1 and could be detected weakly at 6 h of auxin treatment in the WT plant.However, the majority of this transcript induction would likely be due to transformation using the BnARP1 protein coding region without any UTRs and under the control of the 35S promoter.

BnARP1 Affects PIN1 and LOX2 Transcription
To determine the impact of BnARP1 on transcription of auxin genes, three independent Arabidopsis lines harboring high transgene expression levels (BnARP1-OE-1), medium levels (BnARP1-OE-52), and low levels (BnARP1-OE-134) were analyzed by Northern blotting with the following auxin signal transduction and transport genes (Figure 5).BnARP1 potentially could activate any one of the GH3 genes (eg.DWARF IN LIGHT 1, DFL1) to adenylate indole-3-acetic acid (IAA) and reduce free IAA level, shoot growth, and root growth [50], or repress PIN1 trans-membrane proteins in the auxin efflux carrier complex [51], or repress the AUXIN-BINDING PROTEIN 1 (ABP1) receptor  controlling auxin-mediated plant cell expansion [52].BnARP1 could also potentially affect auxin (Aux) response factors (ARFs; directly activating or repressing transcription of target genes), or the Aux/IAA proteins (regulated by auxin to repress ARF function), or the AUXIN RESISTANCE (signal transduction) gene AXR1 important for jasmonate-mediated responses [14] [20].However, five of these six genes (AtDFL1, AtABP1, AtARF1, AtAXR1, and AtIAA1) showed no change in expression with the increased titre of BnARP1 in transgenic Arabidopsis (Figure 9).Only the AtPIN1 gene showed decreased expression in two transgenic Arabidopsis lines BnARP1-OE-52 and -134 with medium-to-low levels of the BnARP1 transgene (Figure 9).This indicates that BnARP1 may participate in auxin transportation.
One study showed that LOX2 and AOS induction by IAA was suppressed in the axr1-24 mutant line, indicating a link between the level of jasmonate synthesis and auxin signaling [20].Surprisingly, LOX2 expression was reduced in all three BnARP1-OE lines (1, 52, and 134) compared with WT (Figure 9).These data suggest that BnARP1 could be involved in regulating the level of jasmonate biosynthesis.

Expression of BnARP1 Improves Arabidopsis Resistance to S. sclerotiorum
In addition to transcript enhancement after flea beetle feeding, drought, and cold, S. sclerotiorum infection also caused very strong increases in BnARP1 in B. napus seedlings (Figure 2).Therefore, bioassays were conducted to determine whether transgenic Arabidopsis expressing this Brassica gene could improve tolerance to S. sclerotiorum.Two BnARP1-OE transgenic lines (No. 134 with weak  trans-gene expression and No. 135 with moderate transgene expression) showed greater seedling survival after S. sclerotiorum infection compared with wild type or empty vector transformed plants (Figure10).The strongest resistance was provided by the BnARP1-OE-135 line (mean of 70% seedling survival).Only BnARP1-OE-1 (with high BnARP1 transgene expression) showed no significant difference in response to S. sclerotiorum.Variation for seedling survival was also higher in individual transgenic lines than in control lines (Figure 10).

Tissue/Stress Specificity and Diversity of the BnARP Gene Family
A survey of expressed genes in a Brassica napus flea beetle damaged leaf EST library revealed an abundance of transcripts for auxin repressed proteins.An expanded search for these genes within our Brassica EST libraries revealed gene family consisting of at least 25 ARP genes (ESTs) that each are differentially expressed in different tissues, developmental stages or in response to different stresses.BnARP gene family is comprised of five sub-groups of genes.The data indicate much larger gene family than previously reported in B. napus or in other Brassica species and support the broad conservation known across higher plants for ARP [25].Individual sequences may be diversified to fit local condi-  tions in individual tissues or under specific stress conditions.However, more closely related ARPs may also have some over-lapping roles or functional redundancy, since our data show that representation of individual BnARP genes is not completely unique to each tissue or stress and both BnARP1 (in this study) and BrARP1overexprssion lines arrest hypocotyl elongation [32].
ARPs are known to be dormancy-associated proteins or induced by abiotic/biotic stress [22] [23] [28] [30].Their induced expression may cause a temporary arrest of plant growth under unfavorable conditions to re-allocate resources into combating stress, as proposed by Lee et al. [32].BnARP1 gene is strongly induced by flea beetle and S. sclerotiorum damage on B. napus leaves and moderately induced by drought and freezing temperatures.The closest BnARP2 orthologue (BrARP1) is also induced by drought and cold and the expression levels of these two orthologues are similar in mature leaves (high), in cotyledon and young leaves (low), and roots (reduced) in transgenic Arabidopsis expression lines [32].A previously reported auxin-repressed gene (GenBank, GU189578) is also induced by drought and high salinity, and appears to be BnARP1, although the scope of its function was only tested in a limited way [31].

BnARP1 May Link Auxin Signaling and JA Synthesis
A lack of apical dominance, a block in primary root extension, and expansion of lateral roots in our Arabidopsis lines confirmed that transgenic expression of induction of LOX2 and AOS was suppressed in the axr1-24 mutant supporting this link between jasmonate and auxin signaling [20].Since BnARP1-OE lines have similar phenotype with axr1-24 mutant lines, and LOX2 gene was suppressed in both types of Arabidopsis lines, it indicates that the BnARP1 gene has opposite function to AtAXR1 gene.Since axr1 lines is more susceptible to the fungus P. irregulare in Arabidopsis demonstrated by Tiryaki and Staswick [20], BnARP1-OE lines are also expected to be more susceptible to the fungus S. sclerotiorum.But our study demonstrated an opposite results in term of resistance to fungus.The reasons for this could be the differentiation of the fungus and BnARP1 is at the different location as ARX1 to link jasmonate and auxin signalling pathways.Unfortunately, our experiment on the impact of exogenous auxin on BnARP1 was limited by the lack of UTRs and other regulatory elements on the BnARP1 binary vector introduced into Arabidopsis.In support of this finding, the RpARP gene from the black locust tree was also not repressed when exogenous auxin was applied to over-expression transgenic plants developed with an RpARP transgene devoid of UTRs [25].Oddly, the BnARP1 expression signal was actually increased as early as 2h after treatment of the transgenic Arabidopsis with exogenous auxin.This could be due in part to hybridization of the BnARP1 probe with the highly similar Arabidopsis ARP1 (Figure S3).However, the applied auxin must also have an enhancing effect on the 35S promoter in these transgenic plants.A TGTCTA element did exist on 35S promoter, which is only one nucleotide different from the auxin response element (AuxRE) TGTCTC [54].This element and many other DNA cis elements exiting on 35S (data not shown) may explain the induction of BnARP1 in overexpression lines by IAA application.The limited test sample for this preliminary experiment must now be expanded to determine whether this phenomenon would occur on a broader set of transgenic lines.

Expression of BnARP1 Improves Tolerance to S. sclerotiorum Infection
Although BnARP1 was nearly unique among their gene family members in their strong response to flea beetle feeding, transgenic Arabidopsis plants expressing BnARP1 did not improve resistance to flea beetle feeding, drought, or cold, although this gene was induced by these conditions in B. napus.In contrast, expression of BnARP1 transgenes in Arabidopsis, strongly improved survival after S. sclerotiorum infection.To BnARP1, its strong induction in stressed B. napus leaves, its effect on growth and failure to improve flea beetle, drought, and cold tolerance, coupled with improved survival after S. sclerotiorum infection in transgenic overexpression Arabidopsis, suggests both an indirect role in slowing plant growth to cope with stress and a direct role in S. sclerotiorum resistance.

Summary of This Study
In summary, ESTs for an Auxin Repressed Protein

Figure 1 .
Figure 1.(a) Phylogenetic amino acid analysis of BnARP gene family (putative from ESTs, all are part of the proteins, except BnARP1) and orthologues (intact proteins) from Arabidopsis, B. rapa and B. oleracea.ARP (auxin repressed protein).Family members used in these phylogenetic trees are shown in the alignment in Figure S1 (in the on-line version); (b) Amino acid sequences alignment of BnARP1-4 and their closest orthologues from B. rapa and B. oleracea.

Figure 2 .
Figure 2. Representative Northern blot analysis of BnARP1 in Brassica napus leaves after different stress applications.Bottom Panel: Ethidium bromide stained rRNA shown as a gel loading control.1) No stress, 2) Crucifer flea beetle feeding, 3) Mechanical wounding, 4) S. sclerotiorum infection, 5) Dehydration, 6) Cold shock 1 h, 7) Cold shock 2 h, 8) Cold shock 7 h.The probe was hybridized with membrane overnight and membrane was exposed to film for 3 h.

Figure 3 .
Figure 3. Representative Northern blot analysis of BnARP1 in Brassica napus cotyledons fed upon by crucifer flea beetles.Bottom panels show ethidium bromide stained rRNA shown as a gel loading control.The probe was hybridized with membrane overnight and membrane was exposed to film for 3 h.

Figure 4 .
Figure 4. Representative Northern blot analysis of BnARP1 expression in specific B. napus tissues and developmental stages.Bottom Panel: Ethidium bromide stained rRNA .Wu et al.
A range of transgenic lines were then verified as having strong Southern blot signals when tested for transgene copy number, although this probe also picked up very weak signals from the native Arabidopsis gene in WT Arabidopsis plants (Figure S2(B)).Representative

Figure 5 .
Figure 5. Representative Northern blot analysis of transgene expression level in seedling leaves of T 2 transgenic Arabidopsis BnARP1-OE lines.

Figure 6 .
Figure 6.Comparison of representative phenotypes of Arabidopsis BnARP1-OE expression lines and wild type.(a) 10 days after germination on selection medium with kanamycin; (b) Primary root growth inhibition after transplanting into MS medium.Root length was measured after 4 d, 7 d, and 14 d of growth.Statistically significant were determined by a Student's t-test.Different letters represent significant differences of the means ± SD (n = 10); (c) Delayed phenotype of transplants at 5 weeks in soil; (d) Primary root growth response on 2, 4-D; (e) lateral root growth response to 2, 4-D for transgenic Arabidopsis line BnARP1-1 OE.Numbers of lateral roots were counted after 10 days growth on MS medium.Statistically significant were determined by a Student's t-test.Different letters represent significant differences of the means ± SD (n = 10).
and BnARP1-OE plants has short primary root and more lateral roots compared to WT (Figure6(a), Figure 6(b) Figure 6(e)), the relationship between root growth inhibition and auxin concentration was investigated in experiments using the synthetic auxin, 2, 4-D and a representative dwarf transgenic Arabidopsis line, BnARP1-OE-1, which had strong transgene expression.Application of a range of 2, 4-D (up to 1.6 μM) to this dwarfed line showed that primary root in L. M. Wu et al.

Figure 7 .
Figure 7. Inhibition of hypocotyl elongation in Arabidopsis BnARP1-OE lines.The BnARP1-OE lines showed reduced hypocotyl elongation with or without 1μM NAA compared to wild type being grown for 4 days in the dark at 22˚C.
lines L. M. Wu et al.

Figure 8 .
Figure 8. Representative Northern blot showing the effect of 2, 4-D application on BnARP1 expression in leaves of transgenic T 2 homozygous Arabidopsis line BnARP1-OE-1.RNAs isolated from leaves of 10-day-old seedlings up to 6 h after spraying with 20 μM 2, 4-D.Numbers below lanes indicate time (h) after treatment was initiated.Radiolabelled BnARP1 whole CDS was hybridized to membranes overnight and the membranes exposed to film for 3 h.Ribosomal RNAs stained with ethidium bromide (EB) indicates equal gel loading of sample RNAs.

Figure 10 .
Figure 10.Seedling survival after S. sclerotiorum infection in Arabidopsis lines expressing BnARP1.S. sclerotiorum inocula were sprayed onto 21-day old plants and leaves assessed after 7 days.WT: wild type, WT + PBI121: transgenic wild type plants with empty PBI121 vector.BnARP1-OE-1, BnARP1-OE-134 and BnARP1-OE-135 are three representative Arabidopsis transgenic lines expressing the BnARP1 gene.Error bars indicate (n = 4).ANOVA and a post-hoc t-test were conducted, such that the different letter indicates significant difference of the means at p < 0.05.
BnARP1 in Arabidopsis can cause an "auxin-depletion" phenotype.Reduction of AtPIN1 transcript levels in two out of three transgenic Arabidopsis BnARP1-OE test lines and reduction in LOX transcripts in all three transgenic BnARP1-OE test lines also suggest that the BnARP1 protein is an auxin-responsive negative regulator that can facilitate a slow-down in plant development by reducing transcription of an auxin transport protein and affecting a JA signalling gene.IAA .Wu et al.

Figure S2 .
Figure S2.Molecular confirmation of the transgenes in Arabidopsis plants.A) Agarose gel showing PCR amplification of transgenes in 3 representative transgenic plants with BnARP1 primers.M: 1 kb plus DNA marker; 1: BnARP1-OE-1; 134: BnARP1-OE-134; 135: BnARP1-OE-135; WT: wild type.The 318 bp bands amplified by BnARP1 primers are BnARP1 CDSs, but the larger band (~500 bp) amplified by BnARP1 primers are part of AT2G33830 genes (see FigureS3).B) Representative Southern analysis of the BnARP1 transgene on restriction enzyme-digested gDNA for wild type and three BnARP1-OE expression lines.Since no restriction cut sites for all three enzymes on BnARP1 CDS probe, it should be only one band on each line, but more than one

Figure S3 .
Figure S3.Alignment of nucleotide sequences of BnARP1 CDS and AT2G33830 DNAs.
L. M.Wu et al.set of ESTs) showed 100% identities with BoARP1, a C genome ancestor protein from Brassica oleracea (Bol036995; http://brassicadb.org/brad/);BnARP2 appeared equally similar to BrARP1, an A genome proteins of Brassica rapa (Bra022955; http://brassicadb.org/brad/);BnARP3 appeared closest to another A damage by the crucifer flea beetle (Phyllotreta cruciferae).Expression of this gene was under different developmental control in B. napus, and it was co-induced in B. napus by flea beetle feeding, S. sclerotiorum infection, drought, and cold.A total of 25 BnARP genes represented in different B. napus stress and development EST libraries indicated larger, diversified families than known earlier.Dwarf phenotypes, primary root growth inhibition, lateral root enhancement, reduced sensitivity to 2, 4-D, and reduced PIN1 and LOX expression in transgenic Arabidopsis expression lines suggest that BnARP1 is an auxin repressor that may prevent auxin transport and supports an interaction between auxin and jasmonate-signaling pathways.The increased survival after S. sclerotiorum infection in transgenic Arabidopsis suggests that BnARP1 may have a direct role in S. sclerotiorum resistance through regulating JA pathway.Therefore, this study also points to a practical use for these BnARP genes and a need for testing their ability to protect Brassica oilseed and vegetable from disease in crop zones with limited growing seasons.
1 (BnARP1) were highly represented (expressed) in a Brassica napus subtractive library developed after L. M. Wu et al. leaf L. M. Wu et al.