Biotic stresses negatively affect canola growth and production. Flea beetle damage and <i> Sclerotinia sclerotiorum (S. sclerotiorum </i> ) infection are two of the worst biotic stresses for canola. <i> Auxin Repressed Proteins (ARPs </i> ) responsive to several abiotic stresses have been reported. However, information about <i> ARPs </i> induced by Flea beetle damage and <i> S. sclerotiorum </i> infection, their roles in biotic stress tolerance are still lacking in canola. ESTs for an <i> Auxin Repressed Protein </i> 1 ( <i> BnARP </i> 1) were highly represented (expressed) in a <i> Brassica napus </i> subtractive library developed after leaf damage by the crucifer flea beetle ( <i> Phyllotreta cruciferae </i> ). Expression of this gene was under different developmental control in <i> B. napus </i> , and it was co-induced in <i> B. napus </i> by flea beetle feeding, <i> S. sclerotiorum </i> infection, drought and cold. A total of 25 <i> BnARP </i> genes were represented in different <i> B. napus </i> 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 <i> PIN </i> 1 and <i> LOX </i> expression in transgenic Arabidopsis expression lines suggest that <i> BnARP </i> 1 is an auxin repressor that prevents auxin transport and supports an interaction between the auxin and jasmonate signalling pathways. And the increased survival after <i> S. sclerotiorum </i> infection in transgenic over-expression Arabidopsis suggests that <i> BnARP </i> 1 could play a role in <i> S. sclerotiorum </i> tolerance through connecting auxin and jasmonate signalling pathways.
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 [
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 [
Auxin repressed proteins (ARPs) are also known. These include development- related ARPs such as the fruit repression SAR5 from strawberry [
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.
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.
Total RNA was isolated with guanidine hydrochloride from undamaged 8-week- old 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 RNAase- free 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 Arabidopsis database (TAIR) and analyzed by bioinformatics according to Gruber et al. [
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
T1 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
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 N2. Total RNA was extracted from tissues as described by Sambrook et al. [
Transgenic Arabidopsis seedling root growth was assayed on T2 homozygous plants growing on Murashige and Skoog (MS) agar plates as described previously [
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. [
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 T2 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 [
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.
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.
A modified S. sclerotiorum infection method was used based on a spray bioassay previously described by Pedras and Ahiahinu [
Analysis of variance was conducted using LSD tests in SAS ver 9.0 [
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 (
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) [
Tissue or Development Stage | |||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Tissue Source | AM | R | S | FB | MFB | MF | VEA | EA | E | C | YL | SF | |||||||||||
aIndividual ARP ESTs | 12(2), 23(1), 24(1) | ND | 14(2) | 5(2) | ND | 21(1), 22(1) | 25(1) | 1(1), 9(1), 10(1) | 8(1), 11(1), 12(1), 13(1) | ND | ND | 1(3) | |||||||||||
Total ESTs | 4844 | 11250 | 2798 | 6014 | 3051 | 6711 | 3680 | 3263 | 5498 | 3838 | 4763 | 1055 | |||||||||||
ARP Representation | 0.08% | 0.00% | 0.07% | 0.03% | 0.00% | 0.03% | 0.03% | 0.09% | 0.07% | 0.00% | 0.00% | 0.28% | |||||||||||
Stress | |||||||||||||||||||||||
Tissue Source | Es1 | Es2 | Cald | Call | DR | DL | M-wls | Fb-dls | Fb-dc | S-is | |||||||||||||
aIndividual ARP ESTs | 1(1), 13(1), 14(3), 15(4), 16(1), 17(1) 18(1) | 2(1), 5(1), 7(1), 8(1), 14(2), 15(3), 19 (2), 20 (1) | 6(1) | ND | ND | ND | ND | 1(17), 2(11), 3(2), 4(1) | 4(1), 7(1), 8(1) | 2(1) | |||||||||||||
Total ESTs | 5551 | 5116 | 6012 | 7914 | 6577 | 5941 | 933 | 1292 | 3762 | 1106 | |||||||||||||
ARP Representation | 0.22% | 0.23% | 0.02% | 0.00% | 0.00% | 0.00% | 0.00% | 2.40% | 0.08% | 0.09% | |||||||||||||
aUnbracketed numbers 1 through 25 indicates 25 unique genes (out of 81 ESTs in total for all the libraries). Bracketed number ( ) shows the frequency of each specific ARP within each EST library (http://brassica.ca and http://brassicagenomics.ca). The coloured highlights indicate the ARPs from the flea beetle damaged leaf subtraction library. ND, not detected. AM: Apical Meristem, R: Root, S: Stem, FB: Flower Bud, MFB: Mature Flower Bud, MF: Mature Flower, VEA: Very Early Anther, EA: Early Anther, E: Embryo, C: Cotyledon, YL: Young Leaf, SF: Senescent Leaf, Es1: Etiolated seedling (vector 1), Es2: Etiolated seedling (vector 2), Cald: Cold-acclimation-leaf (dark), Call: Cold-acclimation-leaf (light), DR: Drought (Root), DL: Drought (Leaf), M-wls: Mechanical-wound leaf subtraction, Fb-dls: Flea beetle-damaged leaf subtraction, Fb-dc: Flea beetle-damaged cotyledon, S-is: S. sclerotiorum-infected stem.
(
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 genome protein BrARP2 (Bra005469; http://brassicadb.org/brad/) and BnARP4 appeared closest to another C genome protein BoARP2 (Bol027300; http://brassicadb.org/brad/) (
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 (
Since cotyledons are critical tissues impacted by flea beetle damage to the canola crop [
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 (
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 (
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.
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
Nearly half of the independently generated T1 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
Lee et al. [
Because auxin plays a role during primary root and lateral root development, and BnARP1-OE plants has short primary root and more lateral roots compared to WT (
hibition was not released, while primary root growth of WT roots was slightly depressed starting at 0.4 μM (
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 [
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 (
controlling auxin-mediated plant cell expansion [
Jasmonate-inducible allene oxide synthase (AOS) and lipoxygenase 2 (LOX2) genes are involved in JA biosynthesis and are elevated by MeJA treatment [
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 (
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 (
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 [
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 [
ARPs are known to be dormancy-associated proteins or induced by abiotic/biotic stress [
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 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 induction of LOX2 and AOS was suppressed in the axr1-24 mutant supporting this link between jasmonate and auxin signaling [
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.
In summary, 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 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.
Limin Wu was the recipient of an NSERC Visiting Fellowship to a Canadian Government Laboratory. This study was supported by a grant to M. Gruber and D. Hegedus from the Genomics Research & Development Initiative (GRDI) of Agriculture and Agri-Food Canada.
Wu, L., Yu, M., Holowachuk, J., Sharpe, A., Lydiate, D., Hegedus, D. and Gruber, M. (2017) Evaluation of a Brassica napus Auxin-Repressed Gene Induced by Flea Beetle Damage and Sclerotinia sclerotiorum Infection. American Journal of Plant Sciences, 8, 1921-1952. https://doi.org/10.4236/ajps.2017.88130