Plant disease resistance is augmented in uzu barley lines modified in the brassinosteroid receptor BRI1
© Ali et al.; licensee BioMed Central Ltd. 2014
Received: 7 April 2014
Accepted: 12 August 2014
Published: 20 August 2014
Brassinosteroid hormones regulate many aspects of plant growth and development. The membrane receptor BRI1 is a central player in the brassinosteroid signaling cascade. Semi-dwarf ‘uzu’ barley carries a mutation in a conserved domain of the kinase tail of BRI1 and this mutant allele is recognised for its positive contribution to both yield and lodging resistance.
Here we show that uzu barley exhibits enhanced resistance to a range of pathogens. It was due to a combination of preformed, inducible and constitutive defence responses, as determined by a combination of transcriptomic and biochemical studies. Gene expression studies were used to determine that the uzu derivatives are attenuated in downstream brassinosteroid signaling. The reduction of BRI1 RNA levels via virus-induced gene silencing compromised uzu disease resistance.
The pathogen resistance of uzu derivatives may be due to pleiotropic effects of BRI1 or the cascade effects of their repressed BR signaling.
KeywordsDisease resistance Brassinosteroid Uzu BRI1 Fusarium
Brassinosteroids (BRs) are a family of hormones, involved in many cellular processes, including cell expansion and division, tissue differentiation, flowering, senescence and responses to abiotic stress ,. BR hormones sequentially bind to the extracellular domains of the leucine rich repeat receptor BR Insensitive 1 (BRI1) and the co-receptor BRI1-associated Kinase 1 (BAK1) . Transphosphorylation between BRI1 and BAK1 activates the former, which in turn leads to a downstream BR signaling cascade . Nakashita et al.  were the first to demonstrate that BR hormones function in disease resistance in both tobacco and rice. Brassinolide (BL) is the end product of the BR biosynthetic pathway and in rice its application enhanced resistance to blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae, respectively. In tobacco, BL induced resistance to Tobacco Mosaic Virus, the bacteria Pseudomonas syringae pv. tabaci and the fungus Oidium sp. Resistance was not associated with accumulation of salicylic acid (SA) and systemic-acquired resistance (SAR) was not involved. Wang  reviewed BR-modulated plant responses to pathogens. In tobacco, virus-induced gene silencing of two homologs of BAK1 (NbSERK3A/B) enhanced susceptibility to the potato blight pathogen Phytophthora infestans, but not to its sister species Phytophthora mirabilis. But BAK1/SERK3 is a multifunctional protein and at least some of its immune functions are independent of BR signaling . It binds to the receptor for bacterial flagellin peptide, FLS2, eliciting PTI. As summarised by Wang , BR activation of BRI1 seems to have two opposite effects on BAK1-mediated FLS2 signaling, and the outcome seems to depend on the relative levels of BR, BRI1, and BAK1. BR signaling inhibits BIN2-mediated degradation of BRI1-EMS-suppressor 1 (BES1); BES1 is then produced and binds to a key defence regulator, AtMYB30, and together they function cooperatively to promote BR target gene expression . BRs may also repress defence gene expression. The BR-activated transcription factor brassinazole-resistant 1 (BZR1) is able to repress the expression of genes such as FLS2 and SNC1 that are directly involved in defence against pathogens ,. But, much remains to be determined regarding how the BR signaling cascade feeds into in plant disease resistance responses.
Uzu barley lines carry a mutation in a highly conserved residue (His-857 to Arg-857) in the kinase domain of the BR receptor protein BRI1 . Recently Goddard et al.  and Chen et al.  showed that introgression of the uzu mutation into barley enhanced resistance to leaf blast disease caused by Magnaporthe grisea, take-all of roots caused by Gaeumannomyces graminis var. tritici, eyespot disease of stems caused by Oculimacula spp. and crown rot disease of the stem caused by Fusarium fungi. Here we investigate the resistance of uzu barley derivatives to more diseases and use a combination of transcriptomic and biochemical studies to determine how these uzu derivatives differ in defences and BR signaling as compared to their parental barley genotypes.
Results and discussion
Uzu enhances resistance to fungal and viral pathogens
Resistance components are constitutive, induced and derepressed
Transcripts that were both pathogen-responsive and differentially regulated (≥1.5 fold) in the uzu derivative of barley cultivar Akashinriki-uzu, as compared to parent type barley line
Microarray data comparisons
Fold induction (uzu vs Aka)¥
Fold induction (uzu + fungus vs uzu + no fungus)¥
Transcripts that were both pathogen up-regulated and up-regulated in uzu versus the parent
Putative ABC transporter
Hypothetical protein B1131B07.13
Putative flavanone 3-hydroxylase
Phosphatidic acid phosphatase beta
Putative peptide transporter
Indole-3-glycerol phosphate synthase
Probable finger protein WZF1
Putative tryptophan synthase α
Putative integral membrane protein
Hypothetical protein P0039A07.26
Nonspecific lipid-transfer protein precursor
Glucan endo-1,3-beta-glucosidase GIII
Pathogenesis-related protein PRB1-3
Thaumatin-like protein TLP7
Thaumatin-like protein TLP4
Putative bromelain-like thiol protaease
Pathogenesis-related protein 1
Pathogenesis-related protein 4
Pathogenesis-related protein 4
Pathogenesis-related protein 4
Glycosyl hydrolase family 17
Pathogen-induced protein WIR1A
Protein id: At4g05020.1
Thaumatin-like protein TLP8
Elicitor inducible gene product EIG-I24
Transcripts that were both pathogen down-regulated and down-regulated in uzu versus the parent
Protein id: At1g29250.1
Putative xyloglucan endotransglycosylase
Translation initiation factor IF-2
BR signaling is repressed in uzu
Virus-induced gene silencing of BRIcomprises disease resistance in uzu
Quantitative RT-PCR gene expression studies validated that VIGS of BRI1 was successful in both Akashinriki-uzu derivative and its parent (Figure 9A). The effect of fungal inoculation after VIGS-mediated silencing was assessed on the 3rd leaf of VIGS-treated seedling. Reducing BRI1 via VIGS enhanced F. culmorum-induced necrosis in leaves of both uzu derivative and parental type plants (Figure 9B). In the absence of BRI1 gene silencing (BSMV:00 treatment), inoculated leaves showed a decrease in infected leaf area, relative to plants subjected to gene silencing (P < 0.01) (Figure 9C). These results suggest that a functional BRI1 is important for disease resistance. Goddard et al.  also reported that a T-DNA insertion in the 5′ untranslated region of BRI1 homolog in Brachypodium distachyon resulted in a similar disease resistance response as observed in the uzu derivatives of barley. More insight into the importance of BRI1 in the disease resistance of uzu requires more in-depth studies of the receptor protein activity in barley derivatives and T-DNA mutants of Brachypodium.
Uzu derivatives are mutated in the kinase domain of BRI1 (this being the only mutation in a conserved domain of the protein). Xu et al.  showed that weak BRI1 mutants of Arabidopsis impaired in kinase activity still retain partial function in plant growth and development, indicating that BRI1 kinase activity is not essential for all activities of this receptor. Uzu resistance may be due to pleiotropic effects of BRI1 on another as yet uncharacterised pathway, indirect effects of the down-regulation of BR signaling or to genetic linkage between BRI1 and a cosegregating resistance gene.
Though the majority of Japanese and Chinese semi-dwarf barley varieties carry the uzu mutation, the stress-intolerance of uzu barley  means that it may not be suited to all climatic conditions. A better understanding of the downstream defence mechanisms might highlight other targets that help control disease in a less environmentally dependent manner. To this end, we are investigating the disease resistance potential of a number of genes up-regulated in uzu in response to F. culmorum.
Plant and microbial material
The barley cultivars (cvs.) Akashinriki and Bowman and their uzu derivatives were kindly provided by Dr. K. Sato, Barley Germplasm Centre, Okayama University, Japan and the John Innes Centre. The uzu derivatives contain the uzu mutant version of the BRI1 gene (GSHO1963), which was derived from Baitori 11, an old Japanese uzu barley. The uzu gene was introduced into barley cv Bowman and cv Akashinriki by sixtime and ninetime backcrossing, respectively . The F. culmorum isolate used in this study was strain FCF 200. The fungus was stored at -70°C and, prior to use, was subcultured onto potato dextrose agar (PDA) (Difco, UK) plates and incubated at 25°C for 5 days. Fungal conidial inoculum was produced in mung bean broth as described previously . Pyrenophora teres f. sp. teres strain N45 was stored at -70°C and, prior to use, were subcultured onto potato dextrose agar (PDA) (Difco, UK) plates and incubated at 25°C for 15 days. The plates containing fungal cultures were scraped and flooded with 0.2% Tween20 solution and the resulting conidial suspension was passed through a double-layer of cheesecloth to obtain the conidial inoculum. The tripartite genome of Barley stripe mosaic virus (BSMV) was maintained within plasmids and RNA generated by in vitro translation was used as inoculum, as previously described by Holzberg et al. .
Fusarium head blight (FHB) experiments
All head blight experiments were conducted in glasshouse chambers at a temperature of 16 - 28°C. Barley cvs. Bowman and Akashinriki and their uzu derivatives were grown and, at mid anthesis, heads were treated with Tween20 (mock treatment) or F. culmorum conidia (1 × 106 spores ml−1 0.2% Tween20), as previously described . Visual disease symptoms were recorded at GS 80 (start of dough development)  based on the percentage of bleached spikelets per head. Treated heads were harvested at growth stage 99. The number of seeds per head and seed weight (g) per head were recorded. Each treatment combination was applied to sixteen plants (2 heads per plant) and the experiment was conducted thrice (February to April 2010, Feb to April 2012 and March to May 2012) in different glass house chambers in a randomized layout.
Fusarium seedling blight (FSB) experiments
Seeds of barley cvs. Bowman and Akashinriki and their uzu derivatives were germinated, grown in a 6 cm diameter pot containing John Innes compost No. 2 (Westland Horticulture, Dun- gannon, UK). The plants were grown in a climate-controlled growth a 12 h light period (700 μmol m−2 s−1) and constant humidity of 85%. Stem bases of 10-day-old seedlings were treated as previously described  with 400 μl of either a F. culmorum conidial suspension (1 × 106 spores ml−1 0.2% Tween20) in 1% agar (Difco Laboratories, Detroit, MI) or 0.2% Tween20 in 1% agar (mock treatment). The stem base samples (4 cm) were harvested at 15 days post-fungal treatment. Seedling blight stem base disease symptoms were scored as the product of lesion length (cm) and lesion colour (lesion colour scale: 0, no disease; 1, very slight brown necrosis; 2, slight/moderate brown necrosis; 3, extensive brown necrosis; 4, extensive black necrosis) . This experiment was conducted thrice, and each time it included three replicate pots (each containing two plants) per treatment combination, arranged in a randomised layout. To analyze gene expression in response to F. culmorum seedling inoculations, similar experiments were conducted on cv. Akashinriki and its uzu derivative, except that samples were flash-frozen in liquid nitrogen and stored at -70°C prior to RNA extraction. Three independent experiments with six replicate pots (each containing two plants) per treatment combination were conducted for microarray analysis (24 h harvest time point) and two independent experiments with three replicate pots (each containing two plants) per treatment combination were conducted for quantitative RT-PCR analysis (24 and 48 h harvest time points).
Net blotch experiments
Seeds of barley cvs. Bowman and Akashinriki and their uzu derivatives were germinated and grown as described above for FSB studies. Foliage of 15 day old barley seedlings were sprayed to runoff with a conidial suspension (4 × 104 spores ml−1 0.2% Tween20 of P. teres f. sp. teres strain N45 or 0.2% Tween20 (mock treatment). The disease score was calculated based on the average infection phenotype of the second and third leaves scored using a 1-10 scale . Results were based on three experiments, all of which included 10 replicate pots (each containing 2 plants) per treatment combination.
Barley stripe mosaic virus experiment
Seeds of barley cvs. Bowman and Akashinriki and their uzu derivatives were germinated and grown as described above for FSB studies, except that experiments were conducted in a contained glasshouse where the temperature was a constant 24°C, and supplemental lighting of 700 μmol m−2 s−1 for 16 h per day was provided. The first leaf of 10 day old seedlings was rub-inoculated with BSMV RNA or with FES buffer (mock treatment) following the protocol described by Scofield et al. . Disease on the third leaf was assessed at 14 days post inoculation, based on the percentage leaf area showing chlorosis. Results were based on two experiments, all of which included 10 replicate pots (each containing 2 plants) per treatment combination.
Detached leaf assay
Seedlings of barley cvs. Bowman and Akashinriki and their uzu derivatives were germinated and grown as described above for FSB studies. The third leaves were harvested from 20 day-old seedlings. Leaf sections (5 cm) were placed on filter paper soaked in 0.08% benzimidazole solution and the upper epidermal layer in the centre of the leaf was surface-wounded by making 4-5 holes using a sterile needle. The damaged leaf area was treated with 5 μl of either 0.2% Tween20 (mock treatment) or F. culmorum conidia (1 × 106 spores ml−1 0.2% Tween20). Leaf samples were photographed 72 h post-inoculation and subjected to 3,3′-diaminobenzidine (DAB) staining to detect ROS formation . Leaf samples were placed in a solution of 1 mg ml−1DAB, and collected for photography after 8 h. Infected leaf area was measured based on the pixel count using Image J software  and the ROS formation was measured based on the total pixel count from 0-100 at a scale of 0-250 and converted to leaf area (2000 pixel = 0.1 cm2).
Seedling composition and epidermal cell morphology
Seedlings of barley cvs. Bowman and Akashinriki and their uzu derivatives were germinated and grown as described above for FSB studies. Stem bases of 10 day old seedlings were treated with either Tween20 in 1% agar or F. culmorum conidia (1 × 106 spores ml−1 0.2% Tween20) and 1% agar. After twenty days, seedlings were cut above the stem base. Leaf sections were treated with absolute alcohol over night at 60°C to remove the chlorophyll and transvers sections were prepared for microscopic study. The remaining green plant material was oven dried for 7-10 days at 55°C and subjected to cellulose and lignin estimation using the methods of Ali et al. . Cellulose and lignin content were determined for three sub-samples per cultivar and were expressed as a percentage of dry weight. Results were based on three experiments, all of which included 10 replicate pots (each containing 2 plants) per treatment combination.
Epibrassinolide (epiBL) treatment experiment
Barley seeds were surface-sterilized with 2% bleach, and kept on Whatman paper in dark at 4°C for 2 days for synchronisation of seed growth. After two days the petri plates were transferred to 25°C degrees in darkness. Three-day-old germinating seedlings were transferred to hydroponic system containing Hoagland’s solution supplemented with 5 μm of Brassinazole (BRZ) in order to inhibit endogenous BR production. The plants were placed in an incubator with continuous light of 700 μmol m−2 s−1 at 25°C. After four days the BRZ solution was replaced by Hoagland’s medium containing 0.2 μm epiBL (in 70% ethanol) and 70% ethanol (mock treatment). Samples were collected from the mock and epiBL treated plants at 12 and 24 h post-treatment and flash frozen in liquid nitrogen prior to RNA extraction.
Total RNA was extracted from the stem base samples using the protocol described by Chang et al. . RNA extracts were DNase1-treated according to manufacturer’s instructions (Invitrogen corp., Carlsbad, CA) and resuspended in diethyl pyrocarbonate (DEPC)-treated water. The quantity of RNA in samples was assessed using an Eppendorf Biophotometer (Eppendorf AG, Hamburg, Germany), according to manufacturer instructions. RNA quality of samples was assessed by estimating the RNA integrity number (RIN) , which averaged > 8, indicating high quality RNA.
Microarray analysis was used to analyse the early effects of F. culmorum on the transcriptome of seedlings of Akashinriki and its uzu derivative (24 h post-fungal inoculation). Microarray production, hybridization, and data analysis were performed following the minimum information about a microarray experiment (MIAME) guidelines for international standardization and quality control of microarray experiments . Microarray analysis was conducted using three composite samples per treatment; composite samples were produced by pooling equal amounts of the total RNA (1 μg) from the five replicate samples per treatment per experiment per time point. Total RNA (1 μg) from each sample was converted to double-stranded cDNA with the Bioarray™ single-round RNA amplification and labeling kit (Enzo life sciences, PA, USA). After second-strand synthesis, the cDNA was purified with the cDNA purification kit (Enzo life sciences). The resulting double-stranded DNA was then used to generate multiple copies of biotinylated cRNA by in vitro transcription with the Bioarray™ highyield™ RNA transcript labeling kit (Enzo life sciences). The A260/280 ratio and yield of each of the cRNAs were determined and the quality of these samples was assessed using an Agilent bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and all exceeded the RNA integrity number threshold of 8. Biotinylated cRNA (10 μg) spiked with biob, bioc, biod and cre (hybridization controls) was hybridized to the Affymetrix barley GeneChip array (Affymetrix, Inc. CA, USA) for 16 hours at 45°C. Following hybridization, all arrays were washed and stained in an Affymetrix GeneChip fluidics station. Stained arrays were scanned with an Affymetrix GeneChip® scanner 3000 (Affymetrix, Inc. CA, USA). Quality checks and data analyses were carried out using affymetrix GeneChip operating software (gcos) and quality reporter. The array data was normalised per chip and per gene. Per chip normalisation is carried out to the median. For per gene normalisation, comparisons were conducted for the three expression values obtained across three biological replicates. Two comparisons were conducted; firstly we compared gene expression in the uzu derivative + fungus, versus Akashinriki + fungus; secondly we compared expression in uzu – fungus versus uzu + fungus. A list of significant probes were generated using a student t-test with a fold-change threshold of ≥ 1.5 and ≤ -1.5 higher transcript with a P-value < 0.0001.
For each probe set, annotations of associated genes/gene homologs were obtained directly from the Affymetrix website (https://www.affymetrix.com/analysis/netaffx/showresults.affx) or by BLASTx analysis against the non-redundant protein database  using the National Center for Biotechnology Information (NCBI) blast resource (http://www.ncbi.nlm.nih.gov). The cut-off value of 10−15 was used as a threshold for the expectation scores (e values), and only homologies with an e-value of less than the threshold were regarded as significant. Whenever, the two descriptions disagreed, the BLASTx description was selected.
Quantitative RT-PCR analysis
Quantitative RT-PCR was used to analyse the expression of transcripts of interest. Reverse transcription (RT) of 1 μg total RNA was conducted as described by Ansari et al. , except that the primer used was oligo dT12-18 (Invitrogen). RT products (25 μl) were diluted to 200 μl and 2.5 μl was PCR-amplified in a 25 μl volume reaction containing 12.5 μl Premix Ex Taq™ (Perfect Real Time) (Takara, Japan) and 100 nM each of forward and reverse transcript-specific primers (Additional file 4: Table S3). PCR reactions were conducted in a Stratagene Mx3000™ quantitative RT-PCR machine (Stratagene, USA) and the programme consisted of 1 cycle of 95°C for 10s, 40 cycles of 95°C for 5 s, 60°C for 30s and 1 cycle of 95°C for 60s. Data were analysed using Stratagene Mx3000™ software (Stratagene, USA). The housekeeping gene used for normalisation of quantitative RT-PCR data was α-tubulin (Affymetrix Contig127_s_at); real-time quantification of target gene and of the housekeeping gene was performed in separate reactions. The threshold cycle (CT) values obtained by quantitative RT-PCR were used to calculate the accumulation of target gene (relative mRNA accumulation), relative to α-tubulin transcript, by 2^-ΔΔCt method, where ΔΔCt = (Ct target gene - Ct α-tubulin) . Results were based on the average obtained for at least two replicate quantitative RT-PCR reactions per sample.
Virus-induced gene silencing (VIGS)
The barley stripe mosaic virus (BSMV)-derived VIGS vectors used in this study consisted of the wild type BSMV ND18 α, β and γ tripartite genome ,. The VIGS fragments and the quantitative RT-PCR assay used to validate VIGS targeted HvBri1 (AB109215.1) on the 3HL chromosome of barley genome, as determined by BLAST analysis against the IPK barley genome database (results not shown). Two independent gene fragments were used for VIGS of HvBri1 and these were amplified from genomic DNA of barley cv. Akashinriki using the primers HvBri1A-F/R or HvBri1B-F/R (Additional file 4: Table S4). PCR reactions were performed with 30 ng of barley genomic DNA, 1 mM each of forward and reverse fragment-specific primers (Additional file 4: Table S4) in a 10 μl reaction containing 0.5U Taq DNA polymerase and 1× PCR buffer (Invitrogen, UK), 1.5 mM MgCl2, and 125 mM of each dNTP. PCR reactions were conducted in a Peltier thermal cycler DNA engine (MJ Research, USA) and the PCR program consisted of an initial denaturation step at 94°C for 2 min, 30 cycles of denaturation 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s and a final extension step at 72°C for 5 min. The amplified silencing fragments were cloned into the pGEM-T vector (pGEM-T Easy cloning kit; Promega, UK). The pGEM-T vectors carrying the silencing fragments were then digested with Pac1 and Sma1. The inserts were purified by gel extraction and then cloned into Pac1 and Sma1 digested γ RNA vector, pSL038-1 . The pSL038-1 plasmids harbouring the silencing fragments were sequenced by Macrogen Inc. (Korea) using the vector-specific primers pGamma-F/R (Additional file 4: Table S4). A BSMV γ RNA construct containing 185 bp-fragment of the barley phytoene desaturase (PDS) gene was used as a positive control for VIGS and has been previously described . The plasmids that contain the BSMV genome α and γ constructs with silencing fragments for PDS and HvBri1A or HvBri1B were linearised with MluI. The plasmid with BSMV β genome was linearised with SpeI. Capped in vitro transcripts were prepared from the linearised plasmids using the mMessage mMachine T7 in vitro transcription kit (Ambion, Austin, TX) following the manufacturer’s protocol. The first leaves of 10-day-old seedlings were rub-inoculated with BSMV constructs following the protocol described by Scofield et al. . Rub inoculations were done with 1:1:1 mixtures of the in vitro transcripts of BSMV α, β and γ RNA (BSMV:00) or derivatives of the γ RNA that contained barley PDS (BSMV:PDS), HvBri1A or HvBri1B fragments. After 14 days the 3rd leaf is taken and made into three segments, one segment flash frozen in liquid N2 and stored at -70°C prior to RNA extraction. Gene silencing was quantified using primers specific to BRI1 and relative to that of the RNA helicase housekeeping gene . The remaining 2 sections were used for the detached leaf Fusarium assay as described above (using a total of 8 leaf sections per Fusarium and 8 per mock Tween20 treatment for each silencing construct). After 3 days symptoms were observed and recorded. The VIGS experiment was conducted three times.
Normal distribution of data sets was determined using the Ryan Joiner test  within Minitab (Minitab release 13.32©, 2000 Minitab Inc.). Non-normally distributed data sets were transformed to fit a normal distribution using the Johnson transformation  within Minitab (Minitab release 13.32©, 2000 Minitab Inc.). The homogeneity of data sets across replicate experiments was confirmed by two-tailed correlation analysis (non-normal data: Spearman Rank; normal data: Pearson product moment) conducted within the Statistical Package for the Social Sciences (SPSS 11.0, SPSS Inc.) (r ≥ 0.798; P = 0.01) . Therefore, data sets from the replicate experiments were pooled for the purposes of further statistical analysis. The significance of treatment effects was analysed within Statistical Package for the Social Sciences (SPSS 11.0, SPSS Inc.) by either (i) normally distributed data - one-way ANOVA with Post Hoc pair wise Least Significance Difference (LSD) comparisons (P = 0.05), or (ii) non-normally-distributed data - the Kruskal-Wallis H test .
Availability of supporting data
The data sets supporting the results of this article are included within the article and its supplementary files.
Provided intellectual and editorial comments: FMD, SSA, PN and SS. Conceived and designed the experiments: SSA and FMD. Performed the experiments: SSA, LRG, GBSK and MK. Analyzed the data: SSA and LRG. Contributed reagents/materials/analysis tools: FMD. Wrote the manuscript: SSA and FMD. All authors read and approved the final manuscript.
This work was supported by the Science Foundation Ireland research fund (IN10/IN.1/B3028) and Department of Agriculture Research Stimulus Grant RSF 07 513. We thank K. Sato, Barley Germplasm Centre, Okayama University, Japan for providing seeds of Akashinriki and its uzu derivative.
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