To determine if damage to foliage by biotic agents, including arthropods, fungi, bacteria and viral pathogens, universally downregulates the expression of genes involved in photosynthesis, we compared transcriptome data from microarray experiments after twenty two different forms of biotic damage on eight different plant species. Transcript levels of photosynthesis light reaction, carbon reduction cycle and pigment synthesis genes decreased regardless of the type of biotic attack. The corresponding upregulation of genes coding for the synthesis of jasmonic acid and those involved in the responses to salicylic acid and ethylene suggest that the downregulation of photosynthesis-related genes was part of a defence response. Analysis of the subcellular targeting of co-expressed gene clusters revealed that the transcript levels of 84% of the genes that carry a chloroplast targeting peptide sequence decreased. The majority of these downregulated genes shared common regulatory elements, such as G-box (CACGTG), T-box (ACTTTG) and SORLIP (GCCAC) motifs. Strong convergence in the response of transcription suggests that the universal downregulation of photosynthesis-related gene expression is an adaptive response to biotic attack. We hypothesize that slow turnover of many photosynthetic proteins allows plants to invest resources in immediate defence needs without debilitating near term losses in photosynthetic capacity.

Nodulation is the result of a mutualistic interaction between legumes and symbiotic soil bacteria (e.g. soybean [Glycine max] and Bradyrhizobium japonicum) initiated by the infection of plant root hair cells by the symbiont. Fewer than 20 plant genes involved in the nodulation process have been functionally characterized. Considering the complexity of the symbiosis, significantly more genes are likely involved. To identify genes involved in root hair cell infection, we performed a large-scale transcriptome analysis of B. japonicum-inoculated and mock-inoculated soybean root hairs using three different technologies: microarray hybridization, Illumina sequencing, and quantitative real-time reverse transcription-polymerase chain reaction. Together, a total of 1,973 soybean genes were differentially expressed with high significance during root hair infection, including orthologs of previously characterized root hair infection-related genes such as NFR5 and NIN. The regulation of 60 genes was confirmed by quantitative real-time reverse transcription-polymerase chain reaction. Our analysis also highlighted changes in the expression pattern of some homeologous and tandemly duplicated soybean genes, supporting their rapid specialization.

Gene expression analysis requires the use of reference genes constitutively expressed independently of tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin, and elongation factor 1-α) are commonly used as reference genes with the assumption that they are uniformly expressed. In many cases, however, this assumption was shown to be incorrect. To provide reliable reference genes in soybean [Glycine max (L.)], we surveyed a set of genes that showed little variation in a nodulation study across a series of soybean microarray experiments. More than 200 putative reference genes were identifi ed. We focused on 18 for further analysis using additional cDNA and Affymetrix arrays and quantitative reverse-transcription polymerase chain reactions. Taken together, these experiments allowed us to test the expression stability of these genes in 130 different conditions, confi rming four soybean genes as new reference genes (annotated as ATP-binding cassette [ABC] transporter, F-box protein family, metalloprotease, and CDPK-related protein kinase). These genes should be useful for normalization of gene expression studies in soybean, an important crop plant.

Atmospheric change studies conducted in free air concentration enrichment (FACE) systems and open-topped chambers have increased understanding of how factors, such as rising CO 2 and O 3 levels, impact the development of plant disease epidemics. Using these systems, plant scientists have been able to study host ⁄ pathogen systems under real-world conditions where variations in multiple environmental parameters impact disease outcomes. Results from these studies are useful for evaluating earlier predictions on plant responses to climate-change parameters and the resulting impacts on plant disease epidemics. Some of these predictions have been verified, whilst others have yet to be tested. Significant interactions among climate-change parameters are highlighting the importance of conducting studies under real-world conditions. The development of molecular and gene expression tools is allowing the fine scale mechanisms responsible for the observed reactions to be determined, and should increase the ability to predict plant disease outcomes under future climatic conditions.

Summary• Transcript profiles in aphid (Aphis glycines)-resistant (cv. Dowling) and -susceptible (cv. Williams 82) soybean (Glycine max) cultivars using soybean cDNA microarrays were investigated.• Large-scale soybean cDNA microarrays representing approx. 18 000 genes or c. 30% of the soybean genome were compared at 6 and 12 h post-application of aphids. In a separate experiment utilizing clip cages, expression of three defense-related genes were examined at 6, 12, 24, 48, and 72 h in both cultivars by quantitative real-time PCR.• One hundred and forty genes showed specific responses for resistance; these included genes related to cell wall, defense, DNA/RNA, secondary metabolism, signaling and other processes. When an extended time period of sampling was investigated, earlier and greater induction of three defense-related genes was observed in the resistant cultivar; however, the induction declined after 24 or 48 h in the resistant cultivar but continued to increase in the susceptible cultivar after 24 h.• Aphid-challenged resistant plants showed rapid differential gene expression patterns similar to the incompatible response induced by avirulent Pseudomonas syringae. Five genes were identified as differentially expressed between the two genotypes in the absence of aphids.

The accumulation of CO2 and O3 in the troposphere alters phytochemistry which in turn influences the interactions between plants and insects. Using microarray analysis of field-grown soybean (Glycine max), we found that the number of transcripts in the leaves affected by herbivory by Japanese beetles (Popillia japonica) was greater when plants were grown under elevated CO2, elevated O3 and the combination of elevated CO2 plus elevated O3 than when grown in ambient atmosphere. The effect of herbivory on transcription diminished strongly with time (<1% of genes were affected by herbivory after 3 weeks), and elevated CO2 interacted more strongly with herbivory than elevated O3. The majority of transcripts affected by elevated O3 were related to antioxidant metabolism. Constitutive levels and the induction by herbivory of key transcripts associated with defence and hormone signalling were down-regulated under elevated CO2; 1-aminocyclopropane-1-carboxylate (ACC) synthase, lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), chalcone synthase (CHS), polyphenol oxidase (PPO) and cysteine protease inhibitor (CystPI) were lower in abundance compared with levels under ambient conditions. By suppressing the ability to mount an effective defence, elevated CO2 may decrease resistance of soybean to herbivory.

P58(IPK) is a cellular inhibitor of the mammalian double-stranded RNA-activated protein kinase (PKR). Here we provide evidence for the existence of its homolog in plants and its role in viral infection at the organism level. Viral infection of P58(IPK)-silenced Nicotiana benthamiana and Arabidopsis knockouts leads to host death. This host cell death is associated with phosphorylation of the alpha subunit of eukaryotic translation initiation factor (eIF-2alpha). Loss of P58(IPK) leads to reduced virus titer, suggesting that wild-type P58(IPK) protein plays an important role in viral pathogenesis. Although our complementation results using mammalian P58(IPK) suggest conservation of the P58(IPK) pathway in plants and animals, its biological significance seems to be different in these two systems. In animals, P58(IPK) is recruited by the influenza virus to limit PKR-mediated innate antiviral response. In plants, P58(IPK) is required by viruses for virulence and therefore functions as a susceptibility factor.

Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation of large quantities of highly pure RNA from plant tissues rich in complex polysaccharides, polyphenolics and waxes. Any contamination of the isolated RNA affects the downstream applications and requires extra cleaning procedures that result in a reduced RNA yield, especially the low molecular weight molecules. The protocol presented here is suitable for isolating high yield and clean total RNA from field-grown plants. Unlike current methods, such as LiCl and TRIZOL, with this new method, the isolated RNA can be used directly for Affymetrix GeneChip labeling or real-time RT-PCR without further purification. This fast and simple protocol provides ready-to-use RNA within 4-5 h after sampling. Additionally, the protocol described here maintains the isolation of small RNA molecules, making it an ideal choice for plant RNA preparation prior to high-throughput sequencing methods to study gene expression.