BACKGROUND
brassinosteroids  are a family of hormones, involved in many cellular processes, including cell expansion and division, tissue differentiation, flowering, senescence and responses to abiotic stress  <cit> .
br hormones sequentially bind to the extracellular domains of the leucine rich repeat receptor br insensitive  <dig>  and the co-receptor bri1-associated kinase  <dig>   <cit> .
transphosphorylation between bri <dig> and bak <dig> activates the former, which in turn leads to a downstream br signaling cascade  <cit> .
nakashita et al.
<cit>  were the first to demonstrate that br hormones function in disease resistance in both tobacco and rice.
brassinolide  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  and systemic-acquired resistance  was not involved.
wang  <cit>  reviewed br-modulated plant responses to pathogens.
in tobacco, virus-induced gene silencing of two homologs of bak <dig>  enhanced susceptibility to the potato blight pathogen phytophthora infestans, but not to its sister species phytophthora mirabilis  <cit> .
but bak1/serk <dig> is a multifunctional protein and at least some of its immune functions are independent of br signaling  <cit> .
it binds to the receptor for bacterial flagellin peptide, fls <dig>  eliciting pti.
as summarised by wang  <cit> , br activation of bri <dig> seems to have two opposite effects on bak1-mediated fls <dig> signaling, and the outcome seems to depend on the relative levels of br, bri <dig>  and bak <dig>  br signaling inhibits bin2-mediated degradation of bri1-ems-suppressor  <dig> ; bes <dig> is then produced and binds to a key defence regulator, atmyb <dig>  and together they function cooperatively to promote br target gene expression  <cit> .
brs may also repress defence gene expression.
the br-activated transcription factor brassinazole-resistant  <dig>  is able to repress the expression of genes such as fls <dig> and snc <dig> that are directly involved in defence against pathogens  <cit> .
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  in the kinase domain of the br receptor protein bri <dig>  <cit> .
recently goddard et al.
<cit>  and chen et al.
<cit>  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
uzu enhances resistance to fungal and viral pathogens
the semi-dwarf uzu derivatives of barley cvs.
akashinriki and bowman were semi-dwarf derived via the introgression of a mutated bri <dig> gene from the old japanese genotype baitori  <dig> and subsequent backcrossing against the parent genotype  <cit> .
pathogenicity tests were used to compare the response of these two semi-dwarf uzu derivatives and their parental barley lines to the obligate pathogen barley stripe mosaic virus , the necrotrophic net blotch pathogen pyrenophora teres and the toxigenic hemibiotrophic fungus fusarium culmorum that causes fusarium head blight .
uzu derivatives were more resistant to all three pathogens as compared to the parental lines.
when compared with their parents, uzu derivatives displayed significantly less symptoms of bsmv, net blotch and fhb disease .
the % of leaf area turned chlorotic due to bsmv was  <dig> and 52% less for the uzu derivatives of akashinriki and bowman, as compared to their respective parent lines .
the severity of net blotch symptoms was 37% less in the akashinriki-uzu derivative and 54% less in bowman-uzu derivative, as compared to the respective parent lines .
the fhb disease development  in the akashinriki-uzu derivative was 65% less than that of the parent line.
in the case of bowman-uzu derivative, infected spikelets were 49% less as compared to the parent line .
fhb disease caused lower reductions in both grain number and weight in the uzu derivatives as compared to parent lines .
fusarium fungi can also attack seedlings and leaves.
seedling blight studies revealed that, in response to f. culmorum, uzu derivative lines of both genotypes developed at least 41% less stem browning and 61% less leaf necrosis than parental lines  .
thus the uzu derivatives carry broad-spectrum disease resistance under controlled conditions, reaffirming and expanding the findings of goddard et al.
<cit> .
this study shows that the disease resistance of uzu derivatives is environment dependent, as the same uzu lines did not show resistance to initial fhb infection in recent uk field trials, albeit the disease pressure was relatively low  <cit> .
goddard et al.
<cit>  recently presented evidence that the uzu derivatives of cvs.
akashinriki and bowman had enhanced resistance to pathogens with a short biotrophic phase or a necrotrophic lifestyle, but not to the biotroph blumeria graminis or to the leaf pathogen ramularia collo-cygni, which has a long asymptomatic phase.figure  <dig>
analysis of the phenotypic effect of barley stripe mosaic virus  on seedling leaves of barley cultivars akashinriki and bowman and their uzu derivatives .
visualisation of the chlorotic stripes on the third leaves at 10 days post-infection.
quantification of the percentage of leaf area showing chlorosis.
bars indicate standard error of mean  .
response of uzu derivatives of barley cultivars akashinriki and bowman  to net blotch disease caused by
pyrenophora teres
f. sp.
teres
.
visualisation of the disease symptoms on seedling leaves at 10 days post-pathogen inoculation.
disease scores based on lesion size  <cit>  on both the second and third leaves at 10 days post-pathogen inoculation.
bars indicate standard error of mean  .
response of uzu derivatives of barley cultivars akashinriki and bowman  to fusarium head blight  disease caused by
fusarium culmorum
.
visualisation of the fhb disease symptoms at growth stage  <dig>   disease symptoms at growth stage  <dig> were quantified as the percentage of infected spikelets per head.
at harvest, the  number of grain per head and the  weight of the grain in each head were determined.
bars indicate standard error of mean  .
resistance components are constitutive, induced and derepressed
microarray analysis of the early fusarium-barley seedling interaction  indicated differential expression of putative defence genes in the akashinriki-uzu derivative compared to the parental line.
comparison  <dig> showed that  <dig> transcripts were ≥  <dig> -fold differentially expressed in f. culmorum-treated akashinriki-uzu derivative as compared to the f. culmorum-treated parent line .
a second microarray comparison delineated  <dig> transcripts as being pathogen-responsive in the uzu derivative  .
looking across both micorarray comparisons, a total of  <dig> transcripts were both more highly expressed in fungus-treated uzu as compared to fungus-treated parent and were also pathogen-responsive in the uzu derivative .
many of these encode putative classical microbe-induced defence and pr proteins .
this includes putative chitinases, endo- <dig> -beta-d-glucosidase and a β- <dig> -glucanase, all of which act synergistically to degrade fungal cell walls  <cit> .
three putative thaumatin-like proteins were both fungal-induced and up-regulated in fungal treated uzu as compared to parent line .
expression of a rice thaumatin-like protein was shown to enhance wheat resistance to fhb  <cit> .
the transcription of indole glycerol phosphate and tryptophan synthase a is indicative of higher auxin production in uzu than the parent and in response to the pathogen.
tryptophan synthase b converts higher indole to tryptophan and this gene, although not pathogen-responsive, was up-regulated in uzu compared to the parent.
multilevel interactions between auxins and brassinosteroids have been reported; based on their study, sakamoto et al.
<cit>  suggested that auxins might control the degree of brassinosteroid perception by regulating the expression of the gene for brassinosteroid receptor.
auxin has been shown to enhance barley resistance to fhb disease  <cit> , although the underpinning mechanisms were not elucidated.
quantitative rt-pcr validated that both a putative wir1a and the classic pr gene pr- <dig> are pathogen –responsive and more highly expressed in uzu derivatives than the parental lines akashinriki and bowman .
wir1a has not been functionally characterised but it is thought to play a role in basal and non-host resistance  <cit> .
wir1a conferred wheat plants with resistance to the powdery mildew fungus blumeria graminis  <cit>  and two variants were mapped with the interval of a wheat genomic locus that confers fhb resistance  <cit> .table  <dig>
transcripts that were both pathogen-responsive and differentially regulated  in the uzu derivative of barley cultivar akashinriki-uzu, as compared to parent type barley line
sequence code
#
microarray data comparisons
gene ontology*
best blastx
±
fold induction
¥
p
-value
fold induction
¥
p
-value
gene/protein id
annotation
e
value
organism
transcripts that were both pathogen up-regulated and up-regulated in uzu versus the parent
oryza sativa
arabidopsis thaliana
oryza sativa
oryza sativa
vigna unguiculata
triticum aestivum
triticum aestivum
oryza sativa
hordeum vulgare
arabidopsis thaliana
triticum aestivum
zea mays
oryza sativa
oryza sativa
malus domestica
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
hordeum vulgare
arabidopsis thaliana
triticum aestivum
pinus taeda
arabidopsis thaliana
hordeum vulgare
nicotiana tabacum
oryza sativa
arabidopsis thaliana
oryza sativa
transcripts that were both pathogen down-regulated and down-regulated in uzu versus the parent
arabidopsis thaliana
arabidopsis thaliana
arabidopsis thaliana
arabidopsis thaliana
oryza sativa
arabidopsis thaliana
#sequences of affymetrix probes used in the microarray analysis were obtained using probe id from http://www.plexdb.org.
±the homologs of barley transcript sequences from the barley database  were determined by blastx analysis against the non-redundant protein database using the ncbi blast resource .
¥fold induction indicated up-regulation of gene in uzu compare to akashinriki or uzu + fungus compare to uzu + no fungus.
*gene ontology was analysed using the web-based tool agrigo http://bioinfo.cau.edu.cn/agrigo/.
analysis of defence and cell wall biosynthesis gene transcript levels in the stem base of
fusarium
-infected seedlings of barley cultivars akashinriki, bowman and their uzu derivatives , as determined by quantitative rt-pcr analysis.
gene represented are  pathogen-induced protein wir1a ,  pathogenesis-related protein  <dig>  ,  cer <dig> protein ,  cellulose synthase-like protein   and  glycine-rich cell wall structural protein  .
bars indicate sem .
looking across both micorarray comparisons, a total of six trancripts were repressed in fungus-treated uzu as compared to the fungus-treated parent and were also pathogen-downregulated .
one of these encoded a putative xyloglucan endotransglycosylase , which catalyses the remodeling of cell walls  <cit> .
the plant cell wall presents a physical barrier to attack by pathogens and cellulose and lignin are amongst the wall constituents that contribute to defence  <cit> .
a preliminary non-replicated microarray analysis at 48 h post-pathogen suggested that various genes involved in the production of wax  and cell wall components  were pathogen-induced in the uzu derivative of cv.
akashinriki .
transcriptomic, biochemical and microscopic analysis were used to investigate the cell size and wall composition in uzu derivatives and the parental lines.
quantitative rt-pcr analyses of uzu derivatives and parents showed that cre <dig>  cellulose synthase and a glycine-rich cell wall structural protein were highly expressed and more pathogen-responsive in uzu derivatives as compared to the parent lines bowman and akashinriki .
biochemical analyses of non-infected uzu did not reveal pathogen-induced increases in the absolute amount of cellulose and lignin , but did confirm that the uzu derivatives have significantly higher cellulose and lignin contents and thicker cell walls than the parental lines .figure  <dig>
the cell wall composition and epidermal cell morphology of leaves of barley cultivars akashinriki and bowman and their uzu derivatives .
twenty days post-stem base treatment the third leaves were removed and cleared for microscopic analysis   the remaining of the green plant tissue was pooled together and analysed for lignin and cellulose content.
bars indicate sem .
further evidence for enhanced cell wall deposition in uzu derivatives was obtained via analysis of bes <dig> transcription.
bes <dig> can associate with upstream elements of most cellulose synthase genes and positively regulates cell wall synthesis  <cit> .
gene expression studies confirmed that seedling leaves from the akashinriki-uzu derivative contained significantly more bes <dig> transcript than those from the parent line akashinriki .
the up-regulation of bes <dig> transcription , up-regulation of four chitinases of which two were pathogen-responsive  and the enhanced ros production during f. culmorum infection of akashinriki-uzu derivative versus the parent line  are all indicative of the enhancement of sa-mediated defence responses  <cit> .
f. culmorum is a hemibiotrophic pathogen, with a short biotrophic phase preceding necrotrophism  <cit> .
concordant with this it has been shown that the host sa dependent defence pathway is activated prior to jasmonic acid  – dependent defence pathways during fusarium infection  <cit> .figure  <dig>
analysis of
bes1
and
bri1
gene transcript levels in barley cultivar akashinriki  and its uzu derivative , as determined by quantitative rt-pcr analysis.
germinating seedlings were transferred to hoaglands solution and the leaf samples are harvested after  <dig> and 72 hours for rna extraction.
brassinazole-resistant- <dig>  .
brassinosteroid insensitive- <dig>  .
bars indicate sem .
recent findings suggest that br may antagonise plant defence signaling via activation of its transcriptional regulator bzr <dig>  upon activation by br, bzr <dig> binds and represses the promoters of various defence-associated genes, including the flagellin receptor fls <dig> and the major r gene snc <dig>  <cit> .
interestingly we found that bzr <dig> homolog of barley was slightly repressed in the akashinriki-uzu-derivative at 48 h post fusarium infection to levels similar to those found in healthy parental seedlings .
this indicates that defences may be slightly derepressed in uzu derivatives.figure  <dig>
analysis of brassinazole-resistant  <dig>  gene transcript levels in the stem base of
fusarium
-infected seedlings of barley cultivars akashinriki, bowman and their uzu derivatives , as determined by quantitative rt-pcr analysis.
rna was extracted from stem bases tissue harvested at either  <dig> or 48 h post-fungal inoculation.
bars indicate sem .
br signaling is repressed in uzu
the literature suggests that the uzu derivative of barley is less responsive to br  <cit> .
we studied the effect of epibrassinolide  treatment on the transcription of genes involved in br signaling.
at the mrna level, the br degradation enzyme gene bas <dig> and the downstream cell wall enzyme gene xet <dig> are both positively regulated by br  <cit> , while the br biosynthesis gene rot <dig> is down-regulated by hormone application  <cit> .
such was the effect of epibl treatment on the transcription of bas <dig> and xet <dig> in the parental cv.
akashinriki, while rot <dig> showed no effect of epibl treatment .
the epibl application induced bas <dig> expression by three-fold by 12 h post treatment in cv.
akashinrki .
but in the uzu derivative, the epibl treatment had no major effect , the trend being for down-regulation of expression of bas <dig> in uzu in response to the hormone .
similar results were obtained for xet <dig> , gene expression being significantly up-regulated in wild type seedlings but significantly down-regulated in uzu by epibl treatment .
we verified that -induced br signaling was repressed in the akashinriki-uzu derivative and thus the enhanced bri <dig> transcription in uzu does not lead to enhanced br signaling.
in both the wild type akashinriki and the uzu derivative, there was no evidence of down- or up-regulation of br biosynthesis, rot <dig> transcription being unresponsive to epibl application .
this suggests that the enhanced resistance of uzu derivatives is not due to br antagonising defence responses or driving enhanced negative cross talk with sa pathways  <cit> .figure  <dig>
transcript levels of
bri1
downstream and brassinosteroid metabolic genes in epibrassinolide  treated seedlings of barley cultivars akashinriki, bowman and their uzu derivatives , as determined by quantitative rt-pcr analysis.
germinating seedlings were treated with hoaglands solution containing 5 μm of the brassinazole for 4 days and then with hoaglands solution plus or minus  <dig>  μm epibl.
gene expression was quantified in samples harvested either  <dig> or 24 h post-treatment.
gene represented are  phyb activation tagged suppressor  <dig> protein, bas <dig> ,
xet  and
rot <dig> .
bars indicate sem .
virus-induced gene silencing of bri <dig> comprises disease resistance in uzu
pathogen-associated molecular pattern triggered immunity  is part of most broad-spectrum resistance/tolerance and brs modulate pti responses through both bak1-dependent and -independent mechanisms  <cit> .
plant disease resistance depends on the relative levels of br, and the receptor proteins bri <dig> and bak <dig>  when the bri <dig> level is low and the bak <dig> level is not rate-limiting, increased br signaling would enhance pti signaling by providing active bak <dig>  <cit> .
however, in uzu derivatives, gene expression studies verified that bri <dig> transcript levels were high as compared to in the parental line akashinriki .
virus-induced gene silencing  was performed in order to investigate the role of bri <dig> in disease resistance.
to help rule out off-target effects in vigs, two independent silencing treatments were performed, each targeting independent fragments of the bri <dig> gene .
empty bsmv vector served as a negative control.
vigs of phytoene desaturase  <cit>  served as a positive control in vigs experiments, resulting in premature bleaching of both akashinriki and uzu derivative plants .
the vigs application buffer fes compromised leaf resistance to f. culmorum.
the uzu/parental type differential observed without fes application  was not observed in the leaf from plants treated with fes .
in both cases, the fusarium disease was assessed on wounded detached sections of the third leaf, the only difference being that the fes applied to the first leaf in the vigs experiments mostly likely induced a systemic wounding response in the vigs experiments.
it is possible, as suggested by goddard et al.
<cit>  that wounding, as occurs in vigs treatment, may swamp the effect of the bri1-related defence responses and may trigger ja signaling  <cit> .figure  <dig>
effect of virus-induced gene silencing  of
bri1
in the barley cultivar akashinriki and its uzu derivatives on
fusarium
infection of seedling leaves as assessed in a detached leaf experiment.
plants were treated with viral application fes buffer, fes plus empty virus  or fes plus virus targeting bri <dig> for silencing .
at 14 days post vigs treatment, 3rd leaf was cleaved off for rna extraction and used for gene expression studies and the detached leaf assay.
bri <dig> expression was quantified.
by 3 days after f. culmorum inoculation, infected area was more evident on gene-silenced as compared to empty virus treated samples of uzu and  this was quantified based on the pixel count using image j software  <cit>  .
bars indicate sem .
quantitative rt-pcr gene expression studies validated that vigs of bri <dig> was successful in both akashinriki-uzu derivative and its parent .
the effect of fungal inoculation after vigs-mediated silencing was assessed on the 3rd leaf of vigs-treated seedling.
reducing bri <dig> via vigs enhanced f. culmorum-induced necrosis in leaves of both uzu derivative and parental type plants .
in the absence of bri <dig> gene silencing , inoculated leaves showed a decrease in infected leaf area, relative to plants subjected to gene silencing  .
these results suggest that a functional bri <dig> is important for disease resistance.
goddard et al.
<cit>  also reported that a t-dna insertion in the 5′ untranslated region of bri <dig> 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 bri <dig> 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.
CONCLUSIONS
uzu derivatives are mutated in the kinase domain of bri <dig> .
xu et al.
<cit>  showed that weak bri <dig> mutants of arabidopsis impaired in kinase activity still retain partial function in plant growth and development, indicating that bri <dig> kinase activity is not essential for all activities of this receptor.
uzu resistance may be due to pleiotropic effects of bri <dig> on another as yet uncharacterised pathway, indirect effects of the down-regulation of br signaling or to genetic linkage between bri <dig> 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  <cit>  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.

 brassinosteroids 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 and the co-receptor bri1-associated kinase. transphosphorylation between bri and bak 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 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 and systemic-acquired resistance was not involved. wang reviewed br-modulated plant responses to pathogens. in tobacco, virus-induced gene silencing of two homologs of bak enhanced susceptibility to the potato blight pathogen phytophthora infestans, but not to its sister species phytophthora mirabilis. but bak1/serk 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, fls eliciting pti. as summarised by wang, br activation of bri seems to have two opposite effects on bak1-mediated fls signaling, and the outcome seems to depend on the relative levels of br, bri and bak br signaling inhibits bin2-mediated degradation of bri1-ems-suppressor; bes is then produced and binds to a key defence regulator, atmyb 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 is able to repress the expression of genes such as fls and snc 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 in the kinase domain of the br receptor protein bri. 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.  uzu enhances resistance to fungal and viral pathogens the semi-dwarf uzu derivatives of barley cvs. akashinriki and bowman were semi-dwarf derived via the introgression of a mutated bri gene from the old japanese genotype baitori and subsequent backcrossing against the parent genotype. pathogenicity tests were used to compare the response of these two semi-dwarf uzu derivatives and their parental barley lines to the obligate pathogen barley stripe mosaic virus, the necrotrophic net blotch pathogen pyrenophora teres and the toxigenic hemibiotrophic fungus fusarium culmorum that causes fusarium head blight. uzu derivatives were more resistant to all three pathogens as compared to the parental lines. when compared with their parents, uzu derivatives displayed significantly less symptoms of bsmv, net blotch and fhb disease. the % of leaf area turned chlorotic due to bsmv was and 52% less for the uzu derivatives of akashinriki and bowman, as compared to their respective parent lines. the severity of net blotch symptoms was 37% less in the akashinriki-uzu derivative and 54% less in bowman-uzu derivative, as compared to the respective parent lines. the fhb disease development in the akashinriki-uzu derivative was 65% less than that of the parent line. in the case of bowman-uzu derivative, infected spikelets were 49% less as compared to the parent line. fhb disease caused lower reductions in both grain number and weight in the uzu derivatives as compared to parent lines. fusarium fungi can also attack seedlings and leaves. seedling blight studies revealed that, in response to f. culmorum, uzu derivative lines of both genotypes developed at least 41% less stem browning and 61% less leaf necrosis than parental lines. thus the uzu derivatives carry broad-spectrum disease resistance under controlled conditions, reaffirming and expanding the findings of goddard et al.. this study shows that the disease resistance of uzu derivatives is environment dependent, as the same uzu lines did not show resistance to initial fhb infection in recent uk field trials, albeit the disease pressure was relatively low. goddard et al. recently presented evidence that the uzu derivatives of cvs. akashinriki and bowman had enhanced resistance to pathogens with a short biotrophic phase or a necrotrophic lifestyle, but not to the biotroph blumeria graminis or to the leaf pathogen ramularia collo-cygni, which has a long asymptomatic phase.figure analysis of the phenotypic effect of barley stripe mosaic virus on seedling leaves of barley cultivars akashinriki and bowman and their uzu derivatives. visualisation of the chlorotic stripes on the third leaves at 10 days post-infection. quantification of the percentage of leaf area showing chlorosis. bars indicate standard error of mean. response of uzu derivatives of barley cultivars akashinriki and bowman to net blotch disease caused by pyrenophora teres f. sp. teres. visualisation of the disease symptoms on seedling leaves at 10 days post-pathogen inoculation. disease scores based on lesion size on both the second and third leaves at 10 days post-pathogen inoculation. bars indicate standard error of mean. response of uzu derivatives of barley cultivars akashinriki and bowman to fusarium head blight disease caused by fusarium culmorum. visualisation of the fhb disease symptoms at growth stage disease symptoms at growth stage were quantified as the percentage of infected spikelets per head. at harvest, the number of grain per head and the weight of the grain in each head were determined. bars indicate standard error of mean. resistance components are constitutive, induced and derepressed microarray analysis of the early fusarium-barley seedling interaction indicated differential expression of putative defence genes in the akashinriki-uzu derivative compared to the parental line. comparison showed that transcripts were ≥  -fold differentially expressed in f. culmorum-treated akashinriki-uzu derivative as compared to the f. culmorum-treated parent line. a second microarray comparison delineated transcripts as being pathogen-responsive in the uzu derivative. looking across both micorarray comparisons, a total of transcripts were both more highly expressed in fungus-treated uzu as compared to fungus-treated parent and were also pathogen-responsive in the uzu derivative. many of these encode putative classical microbe-induced defence and pr proteins. this includes putative chitinases, endo- -beta-d-glucosidase and a β- -glucanase, all of which act synergistically to degrade fungal cell walls. three putative thaumatin-like proteins were both fungal-induced and up-regulated in fungal treated uzu as compared to parent line. expression of a rice thaumatin-like protein was shown to enhance wheat resistance to fhb. the transcription of indole glycerol phosphate and tryptophan synthase a is indicative of higher auxin production in uzu than the parent and in response to the pathogen. tryptophan synthase b converts higher indole to tryptophan and this gene, although not pathogen-responsive, was up-regulated in uzu compared to the parent. multilevel interactions between auxins and brassinosteroids have been reported; based on their study, sakamoto et al. suggested that auxins might control the degree of brassinosteroid perception by regulating the expression of the gene for brassinosteroid receptor. auxin has been shown to enhance barley resistance to fhb disease, although the underpinning mechanisms were not elucidated. quantitative rt-pcr validated that both a putative wir1a and the classic pr gene pr- are pathogen –responsive and more highly expressed in uzu derivatives than the parental lines akashinriki and bowman. wir1a has not been functionally characterised but it is thought to play a role in basal and non-host resistance. wir1a conferred wheat plants with resistance to the powdery mildew fungus blumeria graminis and two variants were mapped with the interval of a wheat genomic locus that confers fhb resistance.table transcripts that were both pathogen-responsive and differentially regulated in the uzu derivative of barley cultivar akashinriki-uzu, as compared to parent type barley line sequence code # microarray data comparisons gene ontology* best blastx ± fold induction ¥ p -value fold induction ¥ p -value gene/protein id annotation e value organism transcripts that were both pathogen up-regulated and up-regulated in uzu versus the parent oryza sativa arabidopsis thaliana oryza sativa oryza sativa vigna unguiculata triticum aestivum triticum aestivum oryza sativa hordeum vulgare arabidopsis thaliana triticum aestivum zea mays oryza sativa oryza sativa malus domestica hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare hordeum vulgare arabidopsis thaliana triticum aestivum pinus taeda arabidopsis thaliana hordeum vulgare nicotiana tabacum oryza sativa arabidopsis thaliana oryza sativa transcripts that were both pathogen down-regulated and down-regulated in uzu versus the parent arabidopsis thaliana arabidopsis thaliana arabidopsis thaliana arabidopsis thaliana oryza sativa arabidopsis thaliana #sequences of affymetrix probes used in the microarray analysis were obtained using probe id from http://www.plexdb.org. ±the homologs of barley transcript sequences from the barley database were determined by blastx analysis against the non-redundant protein database using the ncbi blast resource. ¥fold induction indicated up-regulation of gene in uzu compare to akashinriki or uzu + fungus compare to uzu + no fungus. *gene ontology was analysed using the web-based tool agrigo http://bioinfo.cau.edu.cn/agrigo/. analysis of defence and cell wall biosynthesis gene transcript levels in the stem base of fusarium -infected seedlings of barley cultivars akashinriki, bowman and their uzu derivatives, as determined by quantitative rt-pcr analysis. gene represented are pathogen-induced protein wir1a, pathogenesis-related protein, cer protein, cellulose synthase-like protein and glycine-rich cell wall structural protein. bars indicate sem. looking across both micorarray comparisons, a total of six trancripts were repressed in fungus-treated uzu as compared to the fungus-treated parent and were also pathogen-downregulated. one of these encoded a putative xyloglucan endotransglycosylase, which catalyses the remodeling of cell walls. the plant cell wall presents a physical barrier to attack by pathogens and cellulose and lignin are amongst the wall constituents that contribute to defence. a preliminary non-replicated microarray analysis at 48 h post-pathogen suggested that various genes involved in the production of wax and cell wall components were pathogen-induced in the uzu derivative of cv. akashinriki. transcriptomic, biochemical and microscopic analysis were used to investigate the cell size and wall composition in uzu derivatives and the parental lines. quantitative rt-pcr analyses of uzu derivatives and parents showed that cre cellulose synthase and a glycine-rich cell wall structural protein were highly expressed and more pathogen-responsive in uzu derivatives as compared to the parent lines bowman and akashinriki. biochemical analyses of non-infected uzu did not reveal pathogen-induced increases in the absolute amount of cellulose and lignin, but did confirm that the uzu derivatives have significantly higher cellulose and lignin contents and thicker cell walls than the parental lines.figure the cell wall composition and epidermal cell morphology of leaves of barley cultivars akashinriki and bowman and their uzu derivatives. twenty days post-stem base treatment the third leaves were removed and cleared for microscopic analysis the remaining of the green plant tissue was pooled together and analysed for lignin and cellulose content. bars indicate sem. further evidence for enhanced cell wall deposition in uzu derivatives was obtained via analysis of bes transcription. bes can associate with upstream elements of most cellulose synthase genes and positively regulates cell wall synthesis. gene expression studies confirmed that seedling leaves from the akashinriki-uzu derivative contained significantly more bes transcript than those from the parent line akashinriki. the up-regulation of bes transcription, up-regulation of four chitinases of which two were pathogen-responsive and the enhanced ros production during f. culmorum infection of akashinriki-uzu derivative versus the parent line are all indicative of the enhancement of sa-mediated defence responses. f. culmorum is a hemibiotrophic pathogen, with a short biotrophic phase preceding necrotrophism. concordant with this it has been shown that the host sa dependent defence pathway is activated prior to jasmonic acid – dependent defence pathways during fusarium infection.figure analysis of bes1 and bri1 gene transcript levels in barley cultivar akashinriki and its uzu derivative, as determined by quantitative rt-pcr analysis. germinating seedlings were transferred to hoaglands solution and the leaf samples are harvested after and 72 hours for rna extraction. brassinazole-resistant-. brassinosteroid insensitive-. bars indicate sem. recent findings suggest that br may antagonise plant defence signaling via activation of its transcriptional regulator bzr upon activation by br, bzr binds and represses the promoters of various defence-associated genes, including the flagellin receptor fls and the major r gene snc. interestingly we found that bzr homolog of barley was slightly repressed in the akashinriki-uzu-derivative at 48 h post fusarium infection to levels similar to those found in healthy parental seedlings. this indicates that defences may be slightly derepressed in uzu derivatives.figure analysis of brassinazole-resistant gene transcript levels in the stem base of fusarium -infected seedlings of barley cultivars akashinriki, bowman and their uzu derivatives, as determined by quantitative rt-pcr analysis. rna was extracted from stem bases tissue harvested at either or 48 h post-fungal inoculation. bars indicate sem. br signaling is repressed in uzu the literature suggests that the uzu derivative of barley is less responsive to br. we studied the effect of epibrassinolide treatment on the transcription of genes involved in br signaling. at the mrna level, the br degradation enzyme gene bas and the downstream cell wall enzyme gene xet are both positively regulated by br, while the br biosynthesis gene rot is down-regulated by hormone application. such was the effect of epibl treatment on the transcription of bas and xet in the parental cv. akashinriki, while rot showed no effect of epibl treatment. the epibl application induced bas expression by three-fold by 12 h post treatment in cv. akashinrki. but in the uzu derivative, the epibl treatment had no major effect, the trend being for down-regulation of expression of bas in uzu in response to the hormone. similar results were obtained for xet, gene expression being significantly up-regulated in wild type seedlings but significantly down-regulated in uzu by epibl treatment. we verified that -induced br signaling was repressed in the akashinriki-uzu derivative and thus the enhanced bri transcription in uzu does not lead to enhanced br signaling. in both the wild type akashinriki and the uzu derivative, there was no evidence of down- or up-regulation of br biosynthesis, rot transcription being unresponsive to epibl application. this suggests that the enhanced resistance of uzu derivatives is not due to br antagonising defence responses or driving enhanced negative cross talk with sa pathways.figure transcript levels of bri1 downstream and brassinosteroid metabolic genes in epibrassinolide treated seedlings of barley cultivars akashinriki, bowman and their uzu derivatives, as determined by quantitative rt-pcr analysis. germinating seedlings were treated with hoaglands solution containing 5 μm of the brassinazole for 4 days and then with hoaglands solution plus or minus  μm epibl. gene expression was quantified in samples harvested either or 24 h post-treatment. gene represented are phyb activation tagged suppressor protein, bas, xet and rot. bars indicate sem. virus-induced gene silencing of bri comprises disease resistance in uzu pathogen-associated molecular pattern triggered immunity is part of most broad-spectrum resistance/tolerance and brs modulate pti responses through both bak1-dependent and -independent mechanisms. plant disease resistance depends on the relative levels of br, and the receptor proteins bri and bak when the bri level is low and the bak level is not rate-limiting, increased br signaling would enhance pti signaling by providing active bak. however, in uzu derivatives, gene expression studies verified that bri transcript levels were high as compared to in the parental line akashinriki. virus-induced gene silencing was performed in order to investigate the role of bri in disease resistance. to help rule out off-target effects in vigs, two independent silencing treatments were performed, each targeting independent fragments of the bri gene. empty bsmv vector served as a negative control. vigs of phytoene desaturase served as a positive control in vigs experiments, resulting in premature bleaching of both akashinriki and uzu derivative plants. the vigs application buffer fes compromised leaf resistance to f. culmorum. the uzu/parental type differential observed without fes application was not observed in the leaf from plants treated with fes. in both cases, the fusarium disease was assessed on wounded detached sections of the third leaf, the only difference being that the fes applied to the first leaf in the vigs experiments mostly likely induced a systemic wounding response in the vigs experiments. it is possible, as suggested by goddard et al. that wounding, as occurs in vigs treatment, may swamp the effect of the bri1-related defence responses and may trigger ja signaling.figure effect of virus-induced gene silencing of bri1 in the barley cultivar akashinriki and its uzu derivatives on fusarium infection of seedling leaves as assessed in a detached leaf experiment. plants were treated with viral application fes buffer, fes plus empty virus or fes plus virus targeting bri for silencing. at 14 days post vigs treatment, 3rd leaf was cleaved off for rna extraction and used for gene expression studies and the detached leaf assay. bri expression was quantified. by 3 days after f. culmorum inoculation, infected area was more evident on gene-silenced as compared to empty virus treated samples of uzu and this was quantified based on the pixel count using image j software. bars indicate sem. quantitative rt-pcr gene expression studies validated that vigs of bri was successful in both akashinriki-uzu derivative and its parent. the effect of fungal inoculation after vigs-mediated silencing was assessed on the 3rd leaf of vigs-treated seedling. reducing bri via vigs enhanced f. culmorum-induced necrosis in leaves of both uzu derivative and parental type plants. in the absence of bri gene silencing, inoculated leaves showed a decrease in infected leaf area, relative to plants subjected to gene silencing. these results suggest that a functional bri is important for disease resistance. goddard et al. also reported that a t-dna insertion in the 5′ untranslated region of bri 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 bri 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 bri. xu et al. showed that weak bri mutants of arabidopsis impaired in kinase activity still retain partial function in plant growth and development, indicating that bri kinase activity is not essential for all activities of this receptor. uzu resistance may be due to pleiotropic effects of bri on another as yet uncharacterised pathway, indirect effects of the down-regulation of br signaling or to genetic linkage between bri 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. 
gene expression studies were used to determine that the uzu derivatives are attenuated in downstream brassinosteroid signaling. the pathogen resistance of uzu derivatives may be due to pleiotropic effects of bri or the cascade effects of their repressed br signaling. brassinosteroid hormones regulate many aspects of plant growth and development. it was due to a combination of preformed, inducible and constitutive defence responses, as determined by a combination of transcriptomic and biochemical studies.
 brassinosteroid hormones regulate many aspects of plant growth and development. the membrane receptor bri 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 bri 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 bri rna levels via virus-induced gene silencing compromised uzu disease resistance.  the pathogen resistance of uzu derivatives may be due to pleiotropic effects of bri or the cascade effects of their repressed br signaling. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords disease resistancebrassinosteroiduzubri1fusariumissue-copyright-statement© the author 2014 