BACKGROUND
eucalyptus, a member of the myrtle family, is a genus of woody plants that are keystone ecological species in their natural range in australia and nearby islands.
eucalypt species and hybrids are a valuable international commodity due to their superior growth and wood properties benefiting timber, pulp and paper production.
these trees are also being investigated as a potential lignocellulosic feedstock for biofuel and biomaterials production  <cit> .
long-lived plants such as eucalypts encounter various abiotic and biotic stresses throughout their lifetimes that affect growth and the quality of the wood at rotation age.
an investment in maintaining healthy trees is thus important for ensuring future sustainability of the forestry industry  <cit> .
current disease control strategies such as hygiene practices in nurseries are short-term solutions thus other avenues should be explored to further understand how eucalypts respond during biotic stresses.
the availability of the eucalyptus genome provides an invaluable reservoir to mine for information on various responses such as those activated following an encounter with a pathogen  <cit> .
plant defences have been extensively studied in model organisms such as arabidopsis thaliana and nicotiana spp.
but information about this is limited in eucalypts  <cit> .
from these model systems, it has emerged that plant defence is a multi-faceted and complex process that requires fine-tuning by the host.
perception of a pathogen occurs through receptors in the cell membrane that transduce the signal through various signaling cascades  <cit> .
this transduction results in the initiation of a plethora of mechanisms that alter pathogen proliferation such as the generation of reactive oxygen species , cell wall modifications, hormone signaling and the expression of defence-related proteins  <cit> .
the host needs to be able to tightly regulate these responses as defence is a costly endeavor and these adaptations are usually dependent on the lifestyle of the pathogen.
biotrophic pathogens are restrained through the production of ros and an induction of the salicylic acid  pathway.
however, necrotrophic pathogens can thrive on dead tissue and the production of ros creates a favorable environment for the pathogen which may further promote its proliferation.
defence against necrotrophs then usually involves triggering the ethylene  and jasmonic acid  pathways  <cit> .
chrysoporthe austroafricana is considered a fungal necrotroph that causes the development of stem cankers on e. grandis thereby reducing wood quality, viability and growth  <cit> .
although a devastating pathogen in the late 1990’s, the spread of this fungal pathogen is currently controlled through the vegetative propagation of e. grandis x e. urophylla hybrids.
this interaction between e. grandis and c. austroafricana, can be used as a model and one can exploit this system to expand our understanding of defence in woody species by investigating the responses of a moderately resistant and susceptible host.
in the interaction with c. austroafricana, the e. grandis clone tag <dig> is moderately resistant whilst e. grandis zg <dig> is susceptible, with lesion lengths twice that of tag <dig>  <cit> .
with the availability of the e. grandis genome sequence, this type of study can provide insight into the defence mechanisms employed by the host.
thus the aim of this study was to identify putative defence responses triggered in e. grandis, following challenge by the fungal pathogen c. austroafricana.
this was achieved by profiling known phytohormones and identifying the transcriptional responses activated in the moderately resistant and susceptible hosts through rna-sequencing.
the power of comparing these interactions lies in the ability to deduce basal and induced defence mechanisms that contribute to limiting the spread of the pathogen but also exposes weakness of the host that the pathogen may manipulate.
we find that although there was an overlap between the two hosts in terms of general defence, the moderately resistant host was able to regulate defence responses such as hormone levels that may limit the spread of the disease.
RESULTS
disease progression of c. austroafricana on tag <dig> and zg14
stems of e. grandis ramets of tag <dig> and zg <dig> were inoculated with c. austroafricana and the development of lesions was monitored over the course of six weeks.
measurements taken at 7 days post inoculation  and 3 weeks post inoculation  showed a clear difference in lesion development between tag <dig> and zg <dig> .
the reduced lesion development in tag <dig> was therefore consistent with the classification of this host as moderately resistant according to van heerden et al.
<cit> .figure  <dig>
eucalyptus grandis clones challenged with chrysoporthe austroafricana.
a – lesion development in tag <dig> and zg <dig> at 3 weeks post inoculation.
b – progression of lesion lengths over a 3 week period in tag <dig> and zg <dig>
no lesion development was observed in the mock-inoculated ramets.
while there is indication that wounding would have an effect on a host’s defence responses as observed in model systems  <cit> , the current experiment was designed to use a wounded mock inoculated control.
this type of inoculation closely reflects a natural mode of entry of the fungus in eucalyptus plantations.
while an unwounded control would provide insight into the wound response due to the inoculation procedure, we considered the mock-inoculated control as a more biologically relevant comparison to study the effects of the fungus directly.
hormone profiling highlights changes occurring at  <dig> dpi in tag <dig> that may contribute to resistance
phytohormones form a critical facet of the defence cascade and therefore it was of interest to investigate how these metabolites are influenced in e. grandis under biotic stress.
thus to further evaluate the role of these phytohormone in the interaction with c. austroafricana, we measured ja, sa and gibberellic acid  at various time points in tag <dig> and zg <dig>  this hormone profiling revealed patterns in tag <dig> and zg <dig> that may contribute to resistance or susceptibility respectively.
at the earlier time points  &  <dig> hpi), no significant changes were observed in tag <dig> or zg <dig> with the hormones profiled .
sa was found to occur at significantly higher basal levels in the moderately resistant host tag <dig>  than in the susceptible zg <dig> at all the time points however, following inoculation, sa decreases in tag <dig> at  <dig> dpi but remains constant in zg <dig> .
interestingly at  <dig> dpi, a decrease was also observed for ga in tag <dig> after pathogen challenge but no change was found to occur in zg <dig>  at  <dig> dpi, an increase of ja was observed in tag <dig> and zg <dig>  which also displayed changes in sa and ga having increased and decreased respectively.
concurring with antagonism between sa and ja, the high basal levels of sa in tag <dig> correlated with a lower basal level of ja in tag <dig> than in zg <dig> .figure  <dig> hormone measurements of tag <dig> and zg <dig> over a time series post challenge with c. austroafricana.
tag <dig> mock-inoculated  and tag <dig> inoculated .
zg <dig> mock-inoculated  and zg <dig> inoculated .
error bars are indicative of the standard error of the mean of the biological replicates  for each sample.
significant differences are denoted by different alphabets .
this data alludes to significant changes occurring with the phytohormones at  <dig> dpi in tag  <dig> that are absent in zg <dig> and may contribute to resistance.
additionally there was no significant differences observed in the lesion lengths between the genotypes at  <dig> dpi  and therefore this time point was selected to further investigate additional transcriptional changes that may elucidate other defence mechanisms.
transcriptome profiling of tag <dig> and zg <dig> challenged with c. austroafricana
transcriptome profiling of tag <dig> and zg <dig> at  <dig> dpi  yielded at least  <dig> million total paired ends reads in each sample which all passed quality control assessment .
over 75% of the reads were found to map to the e. grandis genome.
additionally, 2-3% of the reads from the infected samples mapped to the genome of the pathogen, c. austroafricana .
the e. grandis genome currently has  <dig>  predicted protein coding genes  <cit> .
cufflinks analysis of our datasets identified between  <dig>  and  <dig>  expressed genes  .
cuffdiff analyses identified  <dig> and  <dig> significantly de gene models  in tag <dig> and zg <dig> respectively .
the full list of expressed genes can be found in additional file 2: table s <dig>  these gene lists were used for further analyses to elucidate patterns of defence responses emerging from the datasets.figure  <dig> schematic representation of transcriptome profiling conducted with e. grandis challenged with c. austroafricana.
a - lesion development at  <dig> dpi.
b – categories of significantly differentially expressed genes indicating the degree of uniqueness and similarity in the datasets.
c - bar graph representing the number of e. grandis gene models that had corresponding a. thaliana id’s.
over-representation of go terms within the datasets reveals distinct defence responses
to further investigate patterns of the activated defence responses within each host, de genes that had corresponding arabidopsis identities  were analyzed to identify over-representation of gene ontology terms within the three categories: molecular function , cellular component  and biological processes .
this analysis provided an indication of the overall changes occurring within each go category and allows for a broad comparison of the processes occurring in tag <dig> and zg <dig> at the different stages of plant defence.
it also enables the detection of differences in the patterns observed between a resistant and susceptible host indicated by the presence or absence of go terms.
only terms that had a log <dig>  >  <dig> are indicated on the graph and this was applied for all go categories except the biological processes.
for both the up-regulated and down-regulated datasets, the mf and cc categories highlighted the presence of over-represented go terms that were either shared between host responses or unique to a genotype.
for the up-regulated dataset in the mf category, oxidoreductase activity was present in both datasets as genes within this term are generally associated with controlling ros during a defence response .
other terms such as “protein transmembrane transporter activity” was found only in tag <dig>  in the mf down-regulated dataset, there exists a clear difference in “transcription regulator activity” and “chlorophyll binding” between the two hosts.
interestingly, for the cc category, there were more terms unique to the moderately resistant tag <dig> in the up-regulated dataset, while there were more terms unique to the susceptible zg <dig> in the down-regulated dataset .
amongst the shared terms of the up-regulated datasets for cc was the “cell wall” category, which had more over-represented terms in tag <dig> than in zg <dig>
due to the large array of bp responses that were observed, only key processes are highlighted in the figures.
these processes were selected based on known categories of defence that are affected during pathogen interactions.
both hosts contained terms in the up-regulated datasets associated with the phenylpropanoid pathway, response to oxidative stress, secondary metabolic process, lignin metabolic processes, response to ethylene and response to jasmonic acid.
in the zg <dig> up-regulated dataset, there was an over-representation of terms involved in the negative regulation of the et pathway such as “negative regulation of ethylene-mediated signaling pathway” .
this latter term encompasses genes such as ebf1/ <dig> which is involved in suppressing et signaling.
in tag <dig>  the term “defence to fungus, incompatible interaction” was found but did not appear within the zg <dig> dataset.
this corroborates the hypothesis that tag <dig> is moderately resistant and is able to withstand pathogen infection to a degree .figure  <dig> selected biological process go terms that are over-represented in tag <dig> and zg <dig>  a – bingo terms within the up-regulated dataset.
b – bingo terms within the down-regulated dataset for tag <dig>  and zg <dig> .
the y-axis represents the –log <dig> obtained with the benjamini & hochberg false discovery rate  correction analysis.
the x-axis represents the bingo terms within the datasets.
within the down-regulated dataset, there was an over-representation of photosynthesis-related terms in both tag <dig> and zg <dig>  however, zg <dig> also has over-represented terms such as “abscisic acid mediated signaling pathway” and “response of gibberellic acid mediated signaling” that are absent from the tag <dig> dataset .
the candidates within the bp go term categories were subsequently further analyzed to highlight any key distinctions in different metabolic processes.
differences in the regulation of defence responses occur in the two hosts
differentially expressed  gene models from tag <dig> and zg <dig> up – and down-regulated datasets were analyzed with mapman which provides a visual representation of the genes within different cellular processes.
due to the large degree of overlap between the two hosts, the de gene lists were subdivided into sets of genes de in tag <dig> and zg <dig>  those unique to tag <dig>  and those unique to zg <dig> .
defence responses shared between tag <dig> and zg14
from the  <dig> gene models common to both hosts ,  <dig> gene models were found to have corresponding a. thaliana ids.
the gene models that were found to be common amongst the resistant and susceptible interactions correlated with many processes that are affiliated with a defence response.
these processes included up-regulation of genes involved in hormone signaling such as ja and et, an increase in the number of secondary metabolites, changes in the cell wall structure, up-regulation of pathogenesis related proteins as well as the respiratory burst.
although the terms may have been shared between the hosts, the expression levels of the genes associated with these terms were found to be expressed at different levels.
each of these categories also contained genes that were both up- and down-regulated indicating a tight regulation between cellular processes .table  <dig>
genes involved in phytohormone signalling with differential expression  in tag <dig> and zg <dig> following challenge with
c. austroafricana
tair id
euc id
description
abbreviation
tag 5
zg14
sa
ja/et
ga
absence of candidate expression is indicated by n/a.
genes involved in perception and early signal transduction with a differential expression  in tag <dig> and zg <dig> following challenge with
c. austroafricana
tair id
euc id
description
abbreviation
tag 5
zg14
cell wall
ros
signalling
absence of candidates expression is indicated by n/a.
hormone pathways, et and ja, have been extensively shown to be involved in resistance against necrotrophs and thus it was postulated that these pathways would be induced in eucalyptus .
in accordance, genes associated with et that were found to be up-regulated in both hosts include: etr <dig>  aco <dig> and erf <dig>  in terms of de ja genes, lox <dig> and lox <dig> were found to be down-regulated whereas aos and jaz <dig> were found to be up-regulated in tag <dig> and zg <dig>  in addition to hormone signaling, both hosts were found to have a significant number of genes involved in the phenylpropanoid pathway thus potentially implicating this pathway in defence against c. austroafricana.
these included candidates such as phenylalanine ammonia-lyase , o-methyltransferase , caffeoyl coa and cinnamyl-alcohol dehydrogenase.
to facilitate the large energy requirement of a defence response, the host could potentially shunt resources from other metabolic processes such as photosynthesis.
evidence of this diversion was found in the down-regulated datasets of both hosts which highlighted photosystem i and ii go terms .
cell wall degrading enzymes form part of the virulence strategy of pathogens and one of the mechanisms a host employs to resist this is through the methyl esterification of pectins by pectin methylesterase inhibitors   <cit> .
both tag <dig> and zg <dig> exhibited a significant up-regulation of pmei candidates .
damage associated molecular pattern’s  formed during the degradation of the cell wall results in the activation of the signaling cascade.
in plants, the primary activation of ros during a pathogen infection occurs through nadph oxidases, also known as respiratory burst oxidase homologs ,  <cit> .
interestingly atrbohf and atrbohd, both associated with defence, were found to be significantly up-regulated in tag <dig> and zg <dig> at  <dig> pi .
the signaling category revealed that genes involved in calcium signaling was up-regulated in both hosts, but the specific genes encoding for the receptors were different.
of the latter sensors, only ca2+ dependent protein kinases  and calmodulin  were up-regulated in tag <dig> and zg <dig> respectively .
the signal is then transduced through various phosphorylation events to influence transcription factors  and hormone signaling.
in addition to ca2+ signaling, mpk <dig> was found to be significantly up-regulated in zg <dig>  although mpk <dig> was not de in the moderately resistant tag <dig>  the expression level in terms of fpkm values of the gene was comparable between both genotypes following pathogen challenge.
mpk <dig> is known to phosphorylate wrky <dig> and the gene encoding for this tf was found to be significantly up-regulated in both hosts which may mirror the induction of mpk <dig>
regulation of defence processes by tag <dig> may contribute to moderate resistance
despite a degree of overlap in the defence responses that ensue challenge with c. austroafricana, there are marked differences that may influence the outcome of the interaction.
from the  <dig> and  <dig> gene models unique to tag <dig> and zg <dig> respectively,  <dig> and  <dig> gene models had corresponding a. thaliana identities in the respective hosts.
the redox state go category highlighted candidates that were de only in tag <dig> and these include glutathione peroxidase  <dig>  and inositol phosphorylceramide synthase  <dig>  .
within the zg <dig> dataset, the genes categorized under et were different to those expressed within the tag <dig> dataset.
the mapman data confirmed the go ontology results in terms of et whereby, ein <dig> binding f-box <dig>  and ebf <dig> involved in et suppression were found to be unique to zg <dig> and not found in tag <dig> .
another candidate involved in the et biosynthetic pathway, aco <dig>  was found to occur only in the dataset unique to tag <dig> and not zg <dig>  aco <dig> was only de in tag <dig> and the expression of this gene was significantly lower and remained unchanged in zg <dig>
ga is a phytohormone that is well-known for its role in plant development but has also been shown to be involved in pathogen defence.
gasa <dig>  a gene associated with ga signaling was found to be significantly suppressed following infection with c. austroafricana, in both tag <dig> and zg <dig>  this suppression could be due to the increase of ga 2-oxidases  in both hosts which converts active ga to an inactive form  <cit> .
a gene known as scarecrow-like  <dig> , involved in ga regulation is up-regulated in the susceptible zg <dig> but down-regulated in the moderately resistant tag <dig> .
this highlights a time in the defence series at which ga may facilitate resistance in tag <dig> and is supported by a decrease of the hormone at the metabolite level at  <dig> dpi.
the bp go terms highlights the “response to gibberellin stimulus” in the down-regulated dataset of the susceptible interaction.
genes within this go term are involved in the negative regulation of ga signaling such as the della genes rga <dig> and rgl <dig> .
this down-regulation of the della genes in the susceptible host could contribute to higher ga levels than in tag <dig> following inoculation as observed at the metabolite level .
systemic acquired resistance  employs the sa pathway to confer resistance in distal plant tissues.
although sa responsive genes such as pr <dig> were not found to be de at  <dig> dpi, genes associated with sar were found to be distinctive in tag <dig> and zg <dig> .
methyl esterase  <dig>  and plant natriuretric acid  are candidates associated with sar.
atmes <dig> was only up-regulated in tag <dig>  whereas pnp-a was found to be up-regulated in tag <dig> but down-regulated in zg <dig>  thus, tag <dig> may utilize sa to induce an sar response to limit pathogen spread.
therefore although there are cellular responses similar in the two interactions, there are differences between tag <dig> and zg <dig> that may ultimately confer a combinatorial effect resulting in a moderately resistant phenotype.
rt-qpcr analysis reveals a delay in defence responses in the susceptible genotype
candidate genes were selected for rt-qpcr profiling in order to validate the results of the transcriptome analysis.
this selection was based on their transcriptome expression patterns in tag <dig> and zg <dig> at  <dig> dpi as well as their putative role in plant defence .
the genes, 1-aminocyclopropane-1-carboxylate oxidase , inositol phosphorylceramide synthase , plant natriuretic peptide a  and basic pathogenesis related  <dig>  were selected as these candidates showed significant differential expression in tag <dig> but not in zg <dig>  the other two genes, pathogenesis related protein  <dig>  and osmotin  <dig> , are downstream defence products and that were expressed in both hosts but to a significantly higher degree in tag <dig> compared to zg <dig>  all the candidates profiled corroborated with the rna-seq expression patterns.figure  <dig> expression profile of candidate genes from transcriptome profiling post challenge with c. austroafricana.
a – egraco; b – egripcs; c – egrosm34; d – egrpnp-a; e – egrpr1b; f - egrpr <dig>  tag <dig> mock-inoculated  and tag <dig> inoculated .
zg <dig> mock-inoculated  and zg <dig> inoculated .
the y axis represents the relative gene expression ratios as arbitrary units.
error bars are indicative of the standard error of the mean of the biological replicates  for each sample.
significant differences are denoted by different alphabets .
since susceptibility might not only be due to the lack of defence activation but also due to a delayed response of the host to the pathogen, this hypothesis was investigated by profiling the above mentioned candidates at  <dig> dpi in tag <dig> and zg <dig>  interestingly for all the candidates profiled , except egripcs, the level of expression at  <dig> dpi was similar in tag <dig> and zg <dig> for the infected samples.
although the expression level of egripcs was lower in zg <dig> than in tag <dig> at  <dig> dpi, it was significantly higher at  <dig> dpi in zg <dig>  this data provides evidence that zg <dig> host may have a delayed defence response compared to tag <dig>
discussion
modulating hormone signaling may result in the moderate resistance observed in tag5
whilst a degree of overlap exists between the moderately resistant and susceptible host  with regard to the perception and initial activation of defence, the proliferation of the disease caused by c. austroafricana is confined in tag <dig>  thus tag <dig> may modulate defence responses at various levels that collectively contribute to a degree of resistance.
investigating the phytohormone responses activated in the moderately resistant tag <dig> and susceptible zg <dig> suggests a role for various hormones in contributing to defence against c. austroafricana.
et and ja are both known to be involved in defence against necrotrophs and a convergence point at which these pathways interlink is through erf proteins.
whilst the erf <dig> gene is up-regulated in both hosts, the erf <dig> gene is only induced in tag <dig>  erf <dig> is involved in inducing the expression of defence genes that are synchronized by ja and et such as pr <dig> and pr <dig>  <cit> .
supporting this, it was found that although both these pathogenicity genes are up-regulated in tag <dig> and zg <dig>  the level of expression was significantly higher in the moderately resistant host and this increased expression could be attributed to erf <dig>  ja is another signaling pathway that has been implicated in necrotrophic defence, however we did not observe an increase of this phytohormone at the metabolite level following infection until  <dig> dpi.
this may be a contributing factor as to why tag <dig> is moderately resistant and not fully resistant against c. austroafricana.
in a previous study we found that the marker genes of sa, usually associated with defence against biotrophs, were up-regulated in e. grandis following challenge with c. austroafriana  <cit> .
in accordance, this study further elucidates the role of sa through the potential involvement of this hormone in sar.
in the moderately resistant tag <dig>  basal levels of sa were found be significantly higher than the susceptible zg <dig> basal levels at all the investigate time points.
it is possible that higher sa levels may create an sar response in tag <dig> earlier than in the susceptible zg <dig>  the transcript levels of sar markers, atmes <dig> and pnp-a were up-regulated in the moderately resistant tag <dig> at  <dig> dpi thus supporting this notion.
the gene encoding for pnp-a was found to be down-regulated in the susceptible zg <dig>  plant natriuretic peptides are a group of molecules that are involved in sar signaling  <cit> .
although sar originates through sa, this hormone is not the signal that is transduced through the rest of the host.
instead, methyl salicylate  was found to be the systemic signal  <cit> .
mesa is biologically inactive and is converted back to sa in distal tissue to induce sar via methyl esterase   <cit> .
the sar response may be more effective in confining disease spread in the moderately resistant tag <dig> than in the susceptible zg <dig> due to the higher sa levels.
tag <dig> displayed a decrease in ga hormone levels following inoculation whilst the levels remained unchanged in zg <dig> at  <dig> dpi.
gibberelins are a group of hormones that is usually implicated in plant development but have been associated with plant defence.
increased ga levels are associated with enhanced susceptibility towards necrotrophs  <cit> .
ga is under the control of della proteins, part of the gras family proteins that inhibit this signaling pathway  <cit> .
a closely related transcription factor family, designated as scarecrow-like , have been shown to have high similarity to other gras family proteins by containing the conserved vhiid and saw motif  <cit> .
in this study we found scl <dig> to be up-regulated in the susceptible zg <dig> and suppressed in the moderately resistant tag <dig>  scl <dig> was found to modulate della expression through repression thus promoting ga biosynthesis  <cit> , and due to the high similarity with scl <dig>  one can postulate that scl <dig> may be suppressed in tag <dig> to relieve its repression on the della proteins.
the combinatorial effects of an increase in ga2ox and a decrease of scl <dig> in tag <dig> may reduce the levels of ga in this host.
concurrent with this, the up-regulation of scl <dig> may contribute to the suppression of rga and rgl <dig> in zg <dig>
it is intriguing that from our study both sa and ga levels appear to be implicated in resistance as they are both down-regulated in tag <dig> at  <dig> dpi following pathogen challenge.
one possible explanation is the cross-talk between these hormone pathways.
della proteins have also been shown to repress sa signaling  <cit>  and thus the possible relief in della repression through scl <dig> could influence the down-regulation of the sa signaling pathway.
this potential increase in della’s through the down-regulation of scl <dig> in the moderately resistant tag <dig>  coupled with an increase in the expression of wrky <dig>  may stimulate the suppression of sa observed at the metabolite level in tag <dig>
the hormone responses could also be pathogen induced.
it has been shown that pathogens can produce gibberellins as a virulence factor facilitating its own growth and proliferation  <cit> .
a preliminary investigation into the genome of c. austroafricana suggests that this fungus has the ability to produce gibberellins which may manipulate the ga pathway within the host .
ga was first isolated from the pathogenic fungus, gibberella fujikuroi and mutants that were deficient in this hormone production displayed no alteration in development.
thus it has been proposed that pathogens may produce hormones to modulate the defence response of the host.
the production of ga could be employed by pathogens to degrade della proteins therefore allowing for successful colonization  <cit> .
this maybe a possible scenario occurring in the susceptible genotype.
susceptibility could be due to a delay in the expression of defence genes
the ability to detect the pathogen early in defence may afford the host an advantage to activate the correct down-stream response to limit the spread of the pathogen.
susceptibility could either result from the host lacking the required defence genes i.e.
uncertainty in activating the correct response or it could be due to a later activation of the defence genes compared to a resistant host  <cit> .
it is also plausible that both these scenarios could occur simultaneously.
the pattern of a possible delayed defence response was observed in zg <dig> compared to tag <dig>
egraco <dig> and egripcs <dig> were confirmed to be de in moderately resistant tag <dig> but not in the susceptible zg <dig> at  <dig> dpi.
however at  <dig> dpi the expression level of these candidates in zg <dig> is comparable to that of tag <dig>  ipcs <dig> is involved in limiting the spread of pcd which would be beneficial for defence against a necrotrophic pathogen  <cit> .
even in scenarios whereby the expression in tag <dig> was higher compared to zg <dig> at  <dig> dpi, as with egrosm <dig>  egrpr1b and egrpr <dig>  the expression level was similar for both hosts at  <dig> dpi.
thereby this indicates that zg <dig> may have the required artillery to activate an effective defence response, however the timing has a crucial role.
it is evident from the selected genes profiled in this study that tag <dig> may be able to activate a quicker and more robust defence response compared to that of zg <dig> which may contribute to resistance.
CONCLUSIONS
in summary we show that cell wall modifications, fluctuations in the redox state and the combinatorial effects of phytohormones may contribute to moderately resistant phenotype of e. grandis to c. austroafricana.
taken together, the patterns that emerge from the transcriptome profiling suggest that the moderately resistant tag <dig> is able to regulate its defence responses at various phases.
future work would include further investigation into the c. austroafricana genome to explore the pathogenicity mechanism employed by this pathogen.
additionally the data generated in this study illustrates that an integration and coordination of different responses is key in eliciting an effective response in e. grandis and extends our understanding of plant defence in woody species.

 eucalyptus, a member of the myrtle family, is a genus of woody plants that are keystone ecological species in their natural range in australia and nearby islands. eucalypt species and hybrids are a valuable international commodity due to their superior growth and wood properties benefiting timber, pulp and paper production. these trees are also being investigated as a potential lignocellulosic feedstock for biofuel and biomaterials production. long-lived plants such as eucalypts encounter various abiotic and biotic stresses throughout their lifetimes that affect growth and the quality of the wood at rotation age. an investment in maintaining healthy trees is thus important for ensuring future sustainability of the forestry industry. current disease control strategies such as hygiene practices in nurseries are short-term solutions thus other avenues should be explored to further understand how eucalypts respond during biotic stresses. the availability of the eucalyptus genome provides an invaluable reservoir to mine for information on various responses such as those activated following an encounter with a pathogen. plant defences have been extensively studied in model organisms such as arabidopsis thaliana and nicotiana spp. but information about this is limited in eucalypts. from these model systems, it has emerged that plant defence is a multi-faceted and complex process that requires fine-tuning by the host. perception of a pathogen occurs through receptors in the cell membrane that transduce the signal through various signaling cascades. this transduction results in the initiation of a plethora of mechanisms that alter pathogen proliferation such as the generation of reactive oxygen species, cell wall modifications, hormone signaling and the expression of defence-related proteins. the host needs to be able to tightly regulate these responses as defence is a costly endeavor and these adaptations are usually dependent on the lifestyle of the pathogen. biotrophic pathogens are restrained through the production of ros and an induction of the salicylic acid pathway. however, necrotrophic pathogens can thrive on dead tissue and the production of ros creates a favorable environment for the pathogen which may further promote its proliferation. defence against necrotrophs then usually involves triggering the ethylene and jasmonic acid pathways. chrysoporthe austroafricana is considered a fungal necrotroph that causes the development of stem cankers on e. grandis thereby reducing wood quality, viability and growth. although a devastating pathogen in the late 1990’s, the spread of this fungal pathogen is currently controlled through the vegetative propagation of e. grandis x e. urophylla hybrids. this interaction between e. grandis and c. austroafricana, can be used as a model and one can exploit this system to expand our understanding of defence in woody species by investigating the responses of a moderately resistant and susceptible host. in the interaction with c. austroafricana, the e. grandis clone tag is moderately resistant whilst e. grandis zg is susceptible, with lesion lengths twice that of tag. with the availability of the e. grandis genome sequence, this type of study can provide insight into the defence mechanisms employed by the host. thus the aim of this study was to identify putative defence responses triggered in e. grandis, following challenge by the fungal pathogen c. austroafricana. this was achieved by profiling known phytohormones and identifying the transcriptional responses activated in the moderately resistant and susceptible hosts through rna-sequencing. the power of comparing these interactions lies in the ability to deduce basal and induced defence mechanisms that contribute to limiting the spread of the pathogen but also exposes weakness of the host that the pathogen may manipulate. we find that although there was an overlap between the two hosts in terms of general defence, the moderately resistant host was able to regulate defence responses such as hormone levels that may limit the spread of the disease.  disease progression of c. austroafricana on tag and zg14 stems of e. grandis ramets of tag and zg were inoculated with c. austroafricana and the development of lesions was monitored over the course of six weeks. measurements taken at 7 days post inoculation and 3 weeks post inoculation showed a clear difference in lesion development between tag and zg. the reduced lesion development in tag was therefore consistent with the classification of this host as moderately resistant according to van heerden et al..figure eucalyptus grandis clones challenged with chrysoporthe austroafricana. a – lesion development in tag and zg at 3 weeks post inoculation. b – progression of lesion lengths over a 3 week period in tag and zg no lesion development was observed in the mock-inoculated ramets. while there is indication that wounding would have an effect on a host’s defence responses as observed in model systems, the current experiment was designed to use a wounded mock inoculated control. this type of inoculation closely reflects a natural mode of entry of the fungus in eucalyptus plantations. while an unwounded control would provide insight into the wound response due to the inoculation procedure, we considered the mock-inoculated control as a more biologically relevant comparison to study the effects of the fungus directly. hormone profiling highlights changes occurring at dpi in tag that may contribute to resistance phytohormones form a critical facet of the defence cascade and therefore it was of interest to investigate how these metabolites are influenced in e. grandis under biotic stress. thus to further evaluate the role of these phytohormone in the interaction with c. austroafricana, we measured ja, sa and gibberellic acid at various time points in tag and zg this hormone profiling revealed patterns in tag and zg that may contribute to resistance or susceptibility respectively. at the earlier time points & hpi), no significant changes were observed in tag or zg with the hormones profiled. sa was found to occur at significantly higher basal levels in the moderately resistant host tag than in the susceptible zg at all the time points however, following inoculation, sa decreases in tag at dpi but remains constant in zg. interestingly at dpi, a decrease was also observed for ga in tag after pathogen challenge but no change was found to occur in zg at dpi, an increase of ja was observed in tag and zg which also displayed changes in sa and ga having increased and decreased respectively. concurring with antagonism between sa and ja, the high basal levels of sa in tag correlated with a lower basal level of ja in tag than in zg.figure hormone measurements of tag and zg over a time series post challenge with c. austroafricana. tag mock-inoculated and tag inoculated. zg mock-inoculated and zg inoculated. error bars are indicative of the standard error of the mean of the biological replicates for each sample. significant differences are denoted by different alphabets. this data alludes to significant changes occurring with the phytohormones at dpi in tag that are absent in zg and may contribute to resistance. additionally there was no significant differences observed in the lesion lengths between the genotypes at dpi and therefore this time point was selected to further investigate additional transcriptional changes that may elucidate other defence mechanisms. transcriptome profiling of tag and zg challenged with c. austroafricana transcriptome profiling of tag and zg at dpi yielded at least million total paired ends reads in each sample which all passed quality control assessment. over 75% of the reads were found to map to the e. grandis genome. additionally, 2-3% of the reads from the infected samples mapped to the genome of the pathogen, c. austroafricana. the e. grandis genome currently has predicted protein coding genes. cufflinks analysis of our datasets identified between and expressed genes. cuffdiff analyses identified and significantly de gene models in tag and zg respectively. the full list of expressed genes can be found in additional file 2: table s these gene lists were used for further analyses to elucidate patterns of defence responses emerging from the datasets.figure schematic representation of transcriptome profiling conducted with e. grandis challenged with c. austroafricana. a - lesion development at dpi. b – categories of significantly differentially expressed genes indicating the degree of uniqueness and similarity in the datasets. c - bar graph representing the number of e. grandis gene models that had corresponding a. thaliana id’s. over-representation of go terms within the datasets reveals distinct defence responses to further investigate patterns of the activated defence responses within each host, de genes that had corresponding arabidopsis identities were analyzed to identify over-representation of gene ontology terms within the three categories: molecular function, cellular component and biological processes. this analysis provided an indication of the overall changes occurring within each go category and allows for a broad comparison of the processes occurring in tag and zg at the different stages of plant defence. it also enables the detection of differences in the patterns observed between a resistant and susceptible host indicated by the presence or absence of go terms. only terms that had a log  >  are indicated on the graph and this was applied for all go categories except the biological processes. for both the up-regulated and down-regulated datasets, the mf and cc categories highlighted the presence of over-represented go terms that were either shared between host responses or unique to a genotype. for the up-regulated dataset in the mf category, oxidoreductase activity was present in both datasets as genes within this term are generally associated with controlling ros during a defence response. other terms such as “protein transmembrane transporter activity” was found only in tag in the mf down-regulated dataset, there exists a clear difference in “transcription regulator activity” and “chlorophyll binding” between the two hosts. interestingly, for the cc category, there were more terms unique to the moderately resistant tag in the up-regulated dataset, while there were more terms unique to the susceptible zg in the down-regulated dataset. amongst the shared terms of the up-regulated datasets for cc was the “cell wall” category, which had more over-represented terms in tag than in zg due to the large array of bp responses that were observed, only key processes are highlighted in the figures. these processes were selected based on known categories of defence that are affected during pathogen interactions. both hosts contained terms in the up-regulated datasets associated with the phenylpropanoid pathway, response to oxidative stress, secondary metabolic process, lignin metabolic processes, response to ethylene and response to jasmonic acid. in the zg up-regulated dataset, there was an over-representation of terms involved in the negative regulation of the et pathway such as “negative regulation of ethylene-mediated signaling pathway”. this latter term encompasses genes such as ebf1/ which is involved in suppressing et signaling. in tag the term “defence to fungus, incompatible interaction” was found but did not appear within the zg dataset. this corroborates the hypothesis that tag is moderately resistant and is able to withstand pathogen infection to a degree.figure selected biological process go terms that are over-represented in tag and zg a – bingo terms within the up-regulated dataset. b – bingo terms within the down-regulated dataset for tag and zg. the y-axis represents the –log obtained with the benjamini & hochberg false discovery rate correction analysis. the x-axis represents the bingo terms within the datasets. within the down-regulated dataset, there was an over-representation of photosynthesis-related terms in both tag and zg however, zg also has over-represented terms such as “abscisic acid mediated signaling pathway” and “response of gibberellic acid mediated signaling” that are absent from the tag dataset. the candidates within the bp go term categories were subsequently further analyzed to highlight any key distinctions in different metabolic processes. differences in the regulation of defence responses occur in the two hosts differentially expressed gene models from tag and zg up – and down-regulated datasets were analyzed with mapman which provides a visual representation of the genes within different cellular processes. due to the large degree of overlap between the two hosts, the de gene lists were subdivided into sets of genes de in tag and zg those unique to tag and those unique to zg. defence responses shared between tag and zg14 from the gene models common to both hosts, gene models were found to have corresponding a. thaliana ids. the gene models that were found to be common amongst the resistant and susceptible interactions correlated with many processes that are affiliated with a defence response. these processes included up-regulation of genes involved in hormone signaling such as ja and et, an increase in the number of secondary metabolites, changes in the cell wall structure, up-regulation of pathogenesis related proteins as well as the respiratory burst. although the terms may have been shared between the hosts, the expression levels of the genes associated with these terms were found to be expressed at different levels. each of these categories also contained genes that were both up- and down-regulated indicating a tight regulation between cellular processes.table genes involved in phytohormone signalling with differential expression in tag and zg following challenge with c. austroafricana tair id euc id description abbreviation tag 5 zg14 sa ja/et ga absence of candidate expression is indicated by n/a. genes involved in perception and early signal transduction with a differential expression in tag and zg following challenge with c. austroafricana tair id euc id description abbreviation tag 5 zg14 cell wall ros signalling absence of candidates expression is indicated by n/a. hormone pathways, et and ja, have been extensively shown to be involved in resistance against necrotrophs and thus it was postulated that these pathways would be induced in eucalyptus. in accordance, genes associated with et that were found to be up-regulated in both hosts include: etr aco and erf in terms of de ja genes, lox and lox were found to be down-regulated whereas aos and jaz were found to be up-regulated in tag and zg in addition to hormone signaling, both hosts were found to have a significant number of genes involved in the phenylpropanoid pathway thus potentially implicating this pathway in defence against c. austroafricana. these included candidates such as phenylalanine ammonia-lyase, o-methyltransferase, caffeoyl coa and cinnamyl-alcohol dehydrogenase. to facilitate the large energy requirement of a defence response, the host could potentially shunt resources from other metabolic processes such as photosynthesis. evidence of this diversion was found in the down-regulated datasets of both hosts which highlighted photosystem i and ii go terms. cell wall degrading enzymes form part of the virulence strategy of pathogens and one of the mechanisms a host employs to resist this is through the methyl esterification of pectins by pectin methylesterase inhibitors. both tag and zg exhibited a significant up-regulation of pmei candidates. damage associated molecular pattern’s formed during the degradation of the cell wall results in the activation of the signaling cascade. in plants, the primary activation of ros during a pathogen infection occurs through nadph oxidases, also known as respiratory burst oxidase homologs,. interestingly atrbohf and atrbohd, both associated with defence, were found to be significantly up-regulated in tag and zg at pi. the signaling category revealed that genes involved in calcium signaling was up-regulated in both hosts, but the specific genes encoding for the receptors were different. of the latter sensors, only ca2+ dependent protein kinases and calmodulin were up-regulated in tag and zg respectively. the signal is then transduced through various phosphorylation events to influence transcription factors and hormone signaling. in addition to ca2+ signaling, mpk was found to be significantly up-regulated in zg although mpk was not de in the moderately resistant tag the expression level in terms of fpkm values of the gene was comparable between both genotypes following pathogen challenge. mpk is known to phosphorylate wrky and the gene encoding for this tf was found to be significantly up-regulated in both hosts which may mirror the induction of mpk regulation of defence processes by tag may contribute to moderate resistance despite a degree of overlap in the defence responses that ensue challenge with c. austroafricana, there are marked differences that may influence the outcome of the interaction. from the and gene models unique to tag and zg respectively, and gene models had corresponding a. thaliana identities in the respective hosts. the redox state go category highlighted candidates that were de only in tag and these include glutathione peroxidase and inositol phosphorylceramide synthase. within the zg dataset, the genes categorized under et were different to those expressed within the tag dataset. the mapman data confirmed the go ontology results in terms of et whereby, ein binding f-box and ebf involved in et suppression were found to be unique to zg and not found in tag. another candidate involved in the et biosynthetic pathway, aco was found to occur only in the dataset unique to tag and not zg aco was only de in tag and the expression of this gene was significantly lower and remained unchanged in zg ga is a phytohormone that is well-known for its role in plant development but has also been shown to be involved in pathogen defence. gasa a gene associated with ga signaling was found to be significantly suppressed following infection with c. austroafricana, in both tag and zg this suppression could be due to the increase of ga 2-oxidases in both hosts which converts active ga to an inactive form. a gene known as scarecrow-like, involved in ga regulation is up-regulated in the susceptible zg but down-regulated in the moderately resistant tag. this highlights a time in the defence series at which ga may facilitate resistance in tag and is supported by a decrease of the hormone at the metabolite level at dpi. the bp go terms highlights the “response to gibberellin stimulus” in the down-regulated dataset of the susceptible interaction. genes within this go term are involved in the negative regulation of ga signaling such as the della genes rga and rgl. this down-regulation of the della genes in the susceptible host could contribute to higher ga levels than in tag following inoculation as observed at the metabolite level. systemic acquired resistance employs the sa pathway to confer resistance in distal plant tissues. although sa responsive genes such as pr were not found to be de at dpi, genes associated with sar were found to be distinctive in tag and zg. methyl esterase and plant natriuretric acid are candidates associated with sar. atmes was only up-regulated in tag whereas pnp-a was found to be up-regulated in tag but down-regulated in zg thus, tag may utilize sa to induce an sar response to limit pathogen spread. therefore although there are cellular responses similar in the two interactions, there are differences between tag and zg that may ultimately confer a combinatorial effect resulting in a moderately resistant phenotype. rt-qpcr analysis reveals a delay in defence responses in the susceptible genotype candidate genes were selected for rt-qpcr profiling in order to validate the results of the transcriptome analysis. this selection was based on their transcriptome expression patterns in tag and zg at dpi as well as their putative role in plant defence. the genes, 1-aminocyclopropane-1-carboxylate oxidase, inositol phosphorylceramide synthase, plant natriuretic peptide a and basic pathogenesis related were selected as these candidates showed significant differential expression in tag but not in zg the other two genes, pathogenesis related protein and osmotin, are downstream defence products and that were expressed in both hosts but to a significantly higher degree in tag compared to zg all the candidates profiled corroborated with the rna-seq expression patterns.figure expression profile of candidate genes from transcriptome profiling post challenge with c. austroafricana. a – egraco; b – egripcs; c – egrosm34; d – egrpnp-a; e – egrpr1b; f - egrpr tag mock-inoculated and tag inoculated. zg mock-inoculated and zg inoculated. the y axis represents the relative gene expression ratios as arbitrary units. error bars are indicative of the standard error of the mean of the biological replicates for each sample. significant differences are denoted by different alphabets. since susceptibility might not only be due to the lack of defence activation but also due to a delayed response of the host to the pathogen, this hypothesis was investigated by profiling the above mentioned candidates at dpi in tag and zg interestingly for all the candidates profiled, except egripcs, the level of expression at dpi was similar in tag and zg for the infected samples. although the expression level of egripcs was lower in zg than in tag at dpi, it was significantly higher at dpi in zg this data provides evidence that zg host may have a delayed defence response compared to tag discussion modulating hormone signaling may result in the moderate resistance observed in tag5 whilst a degree of overlap exists between the moderately resistant and susceptible host with regard to the perception and initial activation of defence, the proliferation of the disease caused by c. austroafricana is confined in tag thus tag may modulate defence responses at various levels that collectively contribute to a degree of resistance. investigating the phytohormone responses activated in the moderately resistant tag and susceptible zg suggests a role for various hormones in contributing to defence against c. austroafricana. et and ja are both known to be involved in defence against necrotrophs and a convergence point at which these pathways interlink is through erf proteins. whilst the erf gene is up-regulated in both hosts, the erf gene is only induced in tag erf is involved in inducing the expression of defence genes that are synchronized by ja and et such as pr and pr. supporting this, it was found that although both these pathogenicity genes are up-regulated in tag and zg the level of expression was significantly higher in the moderately resistant host and this increased expression could be attributed to erf ja is another signaling pathway that has been implicated in necrotrophic defence, however we did not observe an increase of this phytohormone at the metabolite level following infection until dpi. this may be a contributing factor as to why tag is moderately resistant and not fully resistant against c. austroafricana. in a previous study we found that the marker genes of sa, usually associated with defence against biotrophs, were up-regulated in e. grandis following challenge with c. austroafriana. in accordance, this study further elucidates the role of sa through the potential involvement of this hormone in sar. in the moderately resistant tag basal levels of sa were found be significantly higher than the susceptible zg basal levels at all the investigate time points. it is possible that higher sa levels may create an sar response in tag earlier than in the susceptible zg the transcript levels of sar markers, atmes and pnp-a were up-regulated in the moderately resistant tag at dpi thus supporting this notion. the gene encoding for pnp-a was found to be down-regulated in the susceptible zg plant natriuretic peptides are a group of molecules that are involved in sar signaling. although sar originates through sa, this hormone is not the signal that is transduced through the rest of the host. instead, methyl salicylate was found to be the systemic signal. mesa is biologically inactive and is converted back to sa in distal tissue to induce sar via methyl esterase. the sar response may be more effective in confining disease spread in the moderately resistant tag than in the susceptible zg due to the higher sa levels. tag displayed a decrease in ga hormone levels following inoculation whilst the levels remained unchanged in zg at dpi. gibberelins are a group of hormones that is usually implicated in plant development but have been associated with plant defence. increased ga levels are associated with enhanced susceptibility towards necrotrophs. ga is under the control of della proteins, part of the gras family proteins that inhibit this signaling pathway. a closely related transcription factor family, designated as scarecrow-like, have been shown to have high similarity to other gras family proteins by containing the conserved vhiid and saw motif. in this study we found scl to be up-regulated in the susceptible zg and suppressed in the moderately resistant tag scl was found to modulate della expression through repression thus promoting ga biosynthesis, and due to the high similarity with scl one can postulate that scl may be suppressed in tag to relieve its repression on the della proteins. the combinatorial effects of an increase in ga2ox and a decrease of scl in tag may reduce the levels of ga in this host. concurrent with this, the up-regulation of scl may contribute to the suppression of rga and rgl in zg it is intriguing that from our study both sa and ga levels appear to be implicated in resistance as they are both down-regulated in tag at dpi following pathogen challenge. one possible explanation is the cross-talk between these hormone pathways. della proteins have also been shown to repress sa signaling and thus the possible relief in della repression through scl could influence the down-regulation of the sa signaling pathway. this potential increase in della’s through the down-regulation of scl in the moderately resistant tag coupled with an increase in the expression of wrky may stimulate the suppression of sa observed at the metabolite level in tag the hormone responses could also be pathogen induced. it has been shown that pathogens can produce gibberellins as a virulence factor facilitating its own growth and proliferation. a preliminary investigation into the genome of c. austroafricana suggests that this fungus has the ability to produce gibberellins which may manipulate the ga pathway within the host. ga was first isolated from the pathogenic fungus, gibberella fujikuroi and mutants that were deficient in this hormone production displayed no alteration in development. thus it has been proposed that pathogens may produce hormones to modulate the defence response of the host. the production of ga could be employed by pathogens to degrade della proteins therefore allowing for successful colonization. this maybe a possible scenario occurring in the susceptible genotype. susceptibility could be due to a delay in the expression of defence genes the ability to detect the pathogen early in defence may afford the host an advantage to activate the correct down-stream response to limit the spread of the pathogen. susceptibility could either result from the host lacking the required defence genes i.e. uncertainty in activating the correct response or it could be due to a later activation of the defence genes compared to a resistant host. it is also plausible that both these scenarios could occur simultaneously. the pattern of a possible delayed defence response was observed in zg compared to tag egraco and egripcs were confirmed to be de in moderately resistant tag but not in the susceptible zg at dpi. however at dpi the expression level of these candidates in zg is comparable to that of tag ipcs is involved in limiting the spread of pcd which would be beneficial for defence against a necrotrophic pathogen. even in scenarios whereby the expression in tag was higher compared to zg at dpi, as with egrosm egrpr1b and egrpr the expression level was similar for both hosts at dpi. thereby this indicates that zg may have the required artillery to activate an effective defence response, however the timing has a crucial role. it is evident from the selected genes profiled in this study that tag may be able to activate a quicker and more robust defence response compared to that of zg which may contribute to resistance.  in summary we show that cell wall modifications, fluctuations in the redox state and the combinatorial effects of phytohormones may contribute to moderately resistant phenotype of e. grandis to c. austroafricana. taken together, the patterns that emerge from the transcriptome profiling suggest that the moderately resistant tag is able to regulate its defence responses at various phases. future work would include further investigation into the c. austroafricana genome to explore the pathogenicity mechanism employed by this pathogen. additionally the data generated in this study illustrates that an integration and coordination of different responses is key in eliciting an effective response in e. grandis and extends our understanding of plant defence in woody species. 
this revealed that cell wall modifications and response to oxidative stress form part of the defence responses common to both genotypes, whilst changes in the hormone signaling pathways may contribute to resistance. the ability of a host to fine-tune its defence responses is crucial and the responses identified in this study extends our understanding of plant defence, gained from model systems, to woody perennials. eucalyptus stem cankergibberellic acidhormone signallingplant defencerna-sequencingsalicylic acidissue-copyright-statement© the author 2015 the aim of this study was to characterize the defence response of e. grandis against c. austroafricana. additionally the expression of selected candidate defence response genes was induced earlier in moderately resistant trees than in susceptible trees, supporting the hypothesis that a delayed defence response may occur in the susceptible interaction. however, these trees are challenged by a wide array of biotic stresses during their lifetimes. to further investigate other defence mechanisms at this time point, transcriptome profiling was performed. e. grandis trees are generally susceptible to chrysoporthe austroafricana, a causal agent of stem cankers on eucalypts. hormone profiling of susceptible and moderately resistant clonal e. grandis genotypes indicated a reduction in salicylic acid and gibberellic acid levels at 3 days post inoculation. the eucalyptus grandis reference genome sequence provides a resource to study pest and pathogen defence mechanisms in long-lived woody plants. eucalyptus species and interspecific hybrids exhibit valuable growth and wood properties that make them a highly desirable commodity. we hypothesized that these signaling pathways may facilitate resistance.
 eucalyptus species and interspecific hybrids exhibit valuable growth and wood properties that make them a highly desirable commodity. however, these trees are challenged by a wide array of biotic stresses during their lifetimes. the eucalyptus grandis reference genome sequence provides a resource to study pest and pathogen defence mechanisms in long-lived woody plants. e. grandis trees are generally susceptible to chrysoporthe austroafricana, a causal agent of stem cankers on eucalypts. the aim of this study was to characterize the defence response of e. grandis against c. austroafricana.  hormone profiling of susceptible and moderately resistant clonal e. grandis genotypes indicated a reduction in salicylic acid and gibberellic acid levels at 3 days post inoculation. we hypothesized that these signaling pathways may facilitate resistance. to further investigate other defence mechanisms at this time point, transcriptome profiling was performed. this revealed that cell wall modifications and response to oxidative stress form part of the defence responses common to both genotypes, whilst changes in the hormone signaling pathways may contribute to resistance. additionally the expression of selected candidate defence response genes was induced earlier in moderately resistant trees than in susceptible trees, supporting the hypothesis that a delayed defence response may occur in the susceptible interaction.  the ability of a host to fine-tune its defence responses is crucial and the responses identified in this study extends our understanding of plant defence, gained from model systems, to woody perennials. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords eucalyptus stem cankergibberellic acidhormone signallingplant defencerna-sequencingsalicylic acidissue-copyright-statement© the author 2015 