The authors have declared that no competing interests exist.
Coordinated the study: AMB. Conceived and designed the experiments: PB AMB. Performed the experiments: PB AK DHT. Analyzed the data: PB PW. Contributed reagents/materials/analysis tools: PW AMB. Wrote the paper: PB PW AK AMB.
Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species
The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as
Defence responses and functional networks that were unique and specific to aphid or
Plants are sessile organisms that are unable to escape biotic and abiotic stresses. As a result, they have evolved flexibility in their responses to changing environmental conditions, such as light, drought, temperature, the available nutritional supply and biotic invasion. Different types of biotic invasions, such as insect, bacterial, fungal and viral invasions, represent a severe threat to agricultural production worldwide
Aphids are one of the world’s major insect pests, causing serious economic damage to a range of temperate and tropical crops
A great number of experiments conducted to assess plant responses to different stresses have made substantial contributions to our understanding of the induced defences of plants. However, the comparison of independent experiments and extraction of meaningful information from such comparisons is complicated and difficult in most cases, mainly due to the lack of common standards regarding how to grow plants, conduct expression profile experiments, and finally, how to evaluate the resulting gene expression data
MicroRNAs (microRNAs) are small, non-coding RNAs that play critical roles in post-transcriptional gene regulation and stress-inducible transcriptional regulation in
In this manuscript, we present such an integrated approach to explore the common (general) and attacker-specific defence responses of
Two sets of differentially expressed genes, corresponding to the plant responses to either aphid or bacterial treatment, were created as the outcome of the microarray data analysis. In an attempt to integrate the resulting data with publicly available knowledge extracted from several different databases as well as from published results of other experiments, these two differentially regulated gene sets were subsequently analysed through a set of computational approaches. The following analyses were incorporated into the presented work: an analysis of enriched functional categories or processes; exploration of potential connections between microRNAs and biotic stress-inducible transcriptional regulation during insect and bacterial attack; cross-validation of the aphid- and
To explore the complexity of the transcriptional changes induced by the different examined
Category | No. of Genes | Up- regulated | Down- regulated | No. of TF |
Differentially expressed during Aphid exp. | 4979 | 2803 | 2176 | 303 |
Differentially expressed during |
3199 | 1634 | 1565 | 191 |
1597 | 723 | 688 | 87 | |
Only Aphid | 3382 | 1963 | 1419 | 216 |
Only |
1602 | 842 | 760 | 104 |
In the common set of genes, 186 genes showed opposite expression patterns during the two experiments. Among these genes, 117 were up-regulated under aphid and down-regulated under
In total, 303 transcription factors were found to be affected by the aphid treatment, while 191 transcription factors showed altered expression under
Analysis of overrepresented gene ontologies (GO) in
When this analysis was applied to the list of 1,597 common genes whose expression was affected during both of the experiments, 17 significantly overrepresented categories were identified (some of these categories are shown in
Network representations of enriched GO categories among the genes that were differentially regulated during both experiments. Figure generated from the functionally grouped networks of enriched GO categories among genes whose expression is induced by both the aphid and pathogenic bacterium treatments. GO terms are represented as nodes based on their kappa score (≥0.3); only networks with at least three nodes are represented. The node size indicates the significance of the term’s enrichment. The edges are related to the relationships between the selected terms, which are defined based on the genes that are shared in a similar way. The label of the most significant term is used as the leading group term. Visualisation was conducted using Cytoscape 2.7.0.
Tryptophan-derived indolic compounds, such as indolic glucosinolates (
Localised host programmed cell death is a crucial mechanism through which plants respond to pathogen and insect attack. This phenomenon regulates multiple physiological processes, including terminal differentiation, senescence, and disease resistance
Visualisation of the networks of GO terms based on the aphid-specific responses
Network representations of enriched GO categories among genes that were differentially regulated only during the aphid experiment. Figure generated by ClueGO showing functionally grouped networks of enriched GO categories among genes whose expression was induced only in the aphid experiment. GO terms are represented as nodes based on their kappa score (≥0.3); only networks with at least three nodes are represented. The node size represents the significance of the term’s enrichment. The edges are related to the relationships between the selected terms, which are defined based on the genes that are shared in a similar way. The label for the most significant term is used as the leading group term. Visualisation was conducted using Cytoscape 2.7.0.
Network representations of enriched GO categories among genes that were differentially regulated only during the
To structure the genes present on the
An overview of the transcriptional responses affecting genes coupled to metabolic processes showed that many genes connected to photosynthesis and energy metabolism were down-regulated after
Metabolic pathways associated with the transcriptional changes affecting
A plant’s reaction to biotic stress involves several steps: after the initial signal input from the pathogen, which is recognised by the corresponding receptors (putative R genes), transcription of the cascade associated with the plant defence mechanism is triggered, including changes related to oxidative stress. Inside the cell, signals are transmitted and lead to the production of defence molecules (PR proteins, heat shock proteins and secondary metabolites). A large number of signalling genes were activated during both the aphid and
This figure shows the changes in the expression of biotic stress-responsive genes in
Among the biotic stress-related transcription factors, some
The categories that included most of the induced regulatory genes were TFs, receptor kinases, protein degradation and protein modification. In addition, several genes involved in overrepresented induced biological processes, such as the auxin signalling pathway and autophagy, were included in the regulatory categories
MapMan regulatory overview map showing differences in transcript levels between aphid-specific and
The jasmonic acid signalling pathway is a highly conserved, powerful regulator of plant defence signalling that is activated during infection by various pathogenic microorganisms as well as upon insect attack
Salicylic acid is another stimulator of plant defence responses and is an important trigger of systemic acquired resistance (SAR), resulting in increased defence against a variety of pathogens. Methyl salicylate (MeSA) has been identified as one of the mobile signals required for SAR. MeSA is translocated from the site of infection through the vascular system to distal (systemic) tissues, where it activates specific defence responses. The SAR response results in a complex chain of events and is regulated by various transcription factors. In higher plants, SA can be synthesised from phenylalanine via cinamic acid or from isochorismate. During pathogen attack, SA signalling leads to accumulation of various pathogenesis-related proteins (
Although relatively few genes connected to the biosynthesis of SA and MeSA were found in the obtained datasets, several genes induced by SA were identified. Additionally, isochorismate synthase 1 (
Plants have evolved many secondary metabolites involved in plant defence, which are collectively known as antiherbivory compounds and can be classified into three sub-groups: nitrogen compounds (including alkaloids, cyanogenic glycosides and glucosinolates), terpenoids, and phenolics
Gene ID | Log2 | Description |
At1g52040 | 4.185 |
|
At1g54020 | 3.094 | myrosinase-associated protein, putative |
At5g48180 | 2.293 |
|
At3g19710 | 1.231 | |
At1g16400 | 0.72 |
|
At1g62540 | 0.696 |
|
At5g25980 | 0.487 |
|
At4g13430 | 0.448 |
|
At2g44490 | −0.675 | |
At1g54010 | −0.2 | myrosinase-associated protein, putative |
At1g62570 | 1.56 |
|
Gene ID | Log2 | Description |
At4g03070 | −0.869 |
|
At3g49680 | −1.529 |
|
At3g58990 | −0.648 | aconitase C-terminal domain-containing protein |
At2g43100 | −1.12 | aconitase C-terminal domain-containing protein |
At1g80560 | −0.7 | 3-isopropylmalate dehydrogenase |
At1g31180 | −1.045 | 3-isopropylmalate dehydrogenase |
At4g13770 | −0.415 |
|
At2g31790 | −0.813 | UDP-glucoronosyl/UDP-glucosyl transferase family protein |
At1g18590 | −0.679 |
|
At1g74090 | −0.901 |
|
At1g12140 | 0.313 |
|
At1g65860 | −0.97 |
|
At1g62560 | −0.679 |
|
At4g03060 | −1.248 | |
At5g57220 | 1.527 |
|
At4g31500 | 1.352 |
|
At5g07690 | −1.511 |
|
At5g61420 | −0.707 |
|
At2g33070 | 0.524 |
|
At4g12030 | −0.882 | sodium symporter family protein |
Transcription factors are the key regulators of gene expression changes and, thus, represent important part of a complex regulatory network allowing plants to adjust to changes in their environment
(A) Aphid specific; (B)
The MYB family, which is another large family of TFs characterised by a conserved MYB DNA-binding domain, bind to a variety of different DNA sequences. Among the
We constructed a genetic network of the differentially regulated gene lists using the
Supernode analysis of the differentially expressed common genes using the
Gene ID | microRNA |
At1g53160 |
|
At5g50670 |
|
At3g44860 |
|
At3g44870 |
|
At1g56010 |
|
At5g43780 |
|
At2g33770 |
|
At1g24793 |
|
At1g25054 |
|
References from the literature related to each of the reported microRNA families are provided in
microRNA | Target family |
mir156 |
|
mir157 |
|
mir163 |
|
mir164 |
|
mir395 |
|
mir399 |
|
mir859 |
Among the 3,382 transcripts showing an aphid-specific response, the GO enrichment category ‘response to stimuli (biotic and abiotic stress)’ included 242 stress-responsive genes. Among these genes, 42 are known to exhibit transcription factor activity
A) Among the transcripts showing aphid-specific responses, 42 genes are known to contain microRNA binding sites. Please also refer to
Gene ID | microRNA |
At3g15270 |
|
At2g33810 |
|
At5g43270 |
|
At5g50570 |
|
At5g06100 |
|
At4g30080 |
|
At1g66700 |
|
At1g52150 |
|
At5g37020 |
|
At1g72830 |
|
At5g67180 |
|
At3g15030 |
|
At4g18390 |
|
At3g22890 |
|
At5g53660 |
|
At5g60020 |
|
At1g31280 |
|
At1g12210 |
|
At5g63020 |
|
At1g53290 |
|
At5g42460 |
|
Data retrieved from searches of the published literature and databases. (Refer to
Micro-RNA | Target Gene family |
mir156 |
|
mir157 |
|
mir159 |
|
mir160 |
|
mir163 |
|
mir165 |
|
mir166 |
|
mir167 |
|
mir169 |
|
mir172 |
several genes containing |
mir319 |
|
mir395 |
|
mir397 |
targets several Laccase family members |
mir403 |
|
mir472 |
Several |
mir859 |
Several |
Most, but not all were affected by the aphid treatment.
Among the 1602 transcripts showing
Gene ID | microRNA |
At1g30490 |
|
At1g30210 |
|
At1g53230 |
|
At2g28190 |
|
At1g63360 |
|
At1g24880 |
|
Data retrieved from searches of the published literature and databases. (Refer to
microRNA | Target Gene family |
mir165 |
|
mir166 |
|
mir319 |
|
mir398 |
|
mir472 |
Several |
mir859 |
Several |
Most, but not all were affected by the
The differentially regulated gene sets included many signature transcription factors known for their involvement in stress responses. A co-expression analysis based on a compendium of 69 ATH1 biotic stress experiments, generated using the CORNET tool, showed that many of these TFs have been found to be strongly co-expressed during various biotic stress experiments. From the 66-gene supernode cluster in the common group, the co-expression analysis produced a network of 26 nodes with 25 edges
Co-expression networks generated by CORNET using AtGenExpress biotic stress compendia based on a Pearson’s correlation coefficient threshold ≥0.7. The networks were visualised using Cytoscape2.7.0. Pink-coloured edges represent a strong correlation of ≥0.9, and cyan-coloured edges represent a correlation of ≥0.7 to 0.9.
We generated and analysed data from two different biotic stress experiments conducted in
Categories | Aphid specific | |
Biotic stress signaling processes (up) |
|
|
Biotic stress signaling processes (down) |
|
|
HSPs (up) |
|
|
HSPs(down) |
|
|
Proteolyitc enzymes(up) |
|
|
Proteolyitc enzymes (down) |
|
|
Secondary metabolic(up) |
|
|
Secondary metabolic (down) |
|
|
Cell wall (up) |
|
|
Cell wall (down) |
|
|
TFs (up) |
|
|
TFs(down) |
|
|
Ethylene (up) |
|
|
Ethylene (down) |
|
|
ABA (up) |
|
|
ABA (down) |
|
|
JA (UP) |
|
|
IAA(UP) |
|
|
IAA(Down) |
|
|
SA (UP) | – | BSMT1, UDP-glucoronosyl |
SA(Down) | methyltransferase |
Only those genes with an alias (short annotation name present in TAIR) have been included in this summary table. A complete list of aphid-specific and
This study therefore demonstrates that the integration of heterogeneous publicly available information from multiple databases with experimental results can help plant biologists develop a better understanding of stress-associated processes in plants. Due to logistics and costs we examined only a single time point during the
To overcome the problem of the incompatibility of independent microarray experiments, a genome-wide expression analysis involving 2 different biotic stresses was conducted, in which
The
At 32 days of age (17 days after being transferred to soil), the plants had 8 fully developed leaves. Each plant was infested with 32 wingless aphids (4 per leaf), which were transferred to the leaves with a fine paintbrush. Infested plants and aphid-free controls were maintained in Plexi-glass cylinders, as described previously
A
Total RNA was isolated from cauline leaf tissue from plants from each experiment. Each experiment consisted of four infested samples and four control samples. Total RNA was extracted from 100 mg of cauline leaf material using the RNeasy Plant Minikit (Qiagen, Hilden, Germany) and eluted in 2×50 µl of RNAse-free water. Any residual DNA in the RNA samples was removed by on-column treatment with RNAse-free DNase. The eluted RNA was concentrated to 10–20 µl using a 30 kDa cut-off Microcon spin filter unit (Amicon, Bedford, USA). To protect the RNA from degradation, the RNasin Plus RNase inhibitor (Promega, Madison, USA) was added to a final concentration of 1 unit µl-1. The purity and quantity of the obtained RNA was determined using a Nanodrop ND 1000 instrument (Nanodrop Technologies, Wilmington, DE, USA). RNA integrity was analysed via formaldehyde agarose gel electrophoresis. First-strand cDNA was generated from total RNA (15 µg) using the Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, USA) and oligo dT primers with a 3DNA capture sequence from the 3DNA Array 350TM kit (Genisphere, Hatfield, USA). RNA samples were labelled with either the Cy5-capture primer or Cy3-capture primers (sample dye-swapping). The cDNAs were hybridised to the microarray slides at 58°C using a Slide Booster Hybridisation Station (Advalytix, Brunnthal, Germany) together with Cy3- and Cy5-labelled dendrimers from Genisphere. The slides were washed according to the manufacturers’ instructions (Genisphere and Advalytix).
The microarray slides contained 31811 unique 70-mer oligos with a C6-amino linker, corresponding to a total of 33696 spots, covering 26624 genes. Of these oligos, 29110 were from the Qiagen-Operon
Each dataset obtained from the aphid and
We employed a simple set theory-based operation in R to find common and specific transcriptional responses that occurred in both experiments. To conduct automated GO
The construction of co-expression networks for multiple input genes was conducted using the CORNET tool
The
GO-annotation cytoscape network file (.cys) for common genes.
(CYS)
GO-annotation cytoscape network file (.cys) for aphid-specific genes.
(CYS)
GO-annotation cytoscape network file (.cys) for
(CYS)
MapMan input file for all aphid-responsive genes and corresponding log2-transformed expression values.
(XLS)
MapMan input file for all
(XLS)
Lists of aphid-specific and
(XLS)
List of the 31 heat shock protein (HSP) genes that were differentially expressed only during aphid treatment and their log2-transformed expression values.
(XLS)
List of the proteolyitc enzymes differentially expressed during the aphid and
(XLS)
List of genes related to secondary metabolic processes that were differentially regulated during the aphid and
(XLS)
List of genes involved in cell wall precursor synthesis that were differentially regulated during the aphid and
(XLS)
A comparative list of the differentially expressed genes (aphid-specific and
(XLS)
Details of all of the TFs that were differentially expressed during the aphid and Pseudmonas treatments.
(XLS)
The authors thank Torfinn Sparstad for excellent technical assistance.