Overexpression of a soybean 4-coumaric acid: coenzyme A ligase (GmPI4L) enhances resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot of soybean (Glycine max (L.) Merr.) caused by Phytophthora sojae is a destructive disease worldwide. The enzyme 4-coumarate: CoA ligase (4CL) has been extensively studied with regard to plant responses to pathogens. However, the molecular mechanism of the response of soybean 4CL to P. sojae remains unclear. In a previous study, a highly upregulated 4CL homologue was characterised through suppressive subtractive hybridisation library and cDNA microarrays, in the resistant soybean cultivar ‘Suinong 10’ after infection with P. sojae race 1. Here, we isolated the full-length EST, and designated as GmPI4L (P. sojae-inducible 4CL gene) in this study, which is a novel member of the soybean 4CL gene family. GmPI4L has 34–43% over all amino acid sequence identity with other plant 4CLs. Overexpression of GmPI4L enhances resistance to P. sojae in transgenic soybean plants. The GmPI4L is located in the cell membrane when transiently expressed in Arabidopsis protoplasts. Further analyses showed that the contents of daidzein, genistein, and the relative content of glyceollins are significantly increased in overexpression GmPI4L soybeans. Taken together, these results suggested that GmPI4L plays an important role in response to P. sojae infection, possibly by enhancing the content of glyceollins, daidzein, and genistein in soybean.

Identification of drought-inducible regulatory factors in Lablab purpureus by a comparative genomic approach

Physiological indices of two lines of hyacinth bean (Lablab purpureus (L.) Sweet), MEIDOU 2012 and NANHUI 23, were compared during a progressive course of water-withholding treatments. MEIDOU 2012 was shown to be a superior drought-tolerant genotype. A suppressive subtractive hybridisation library was then constructed by using drought-stressed MEIDOU 2012 roots and sequenced to screen for transcripts with differential abundance variations. In total, 2792 unigenes were assembled from 4064 drought-induced expressed sequence tags. Comparative analysis with other legume genomic sequences, including soybean (Glycine max (L.) Merr.), common bean (Phaseolus vulgaris L.) and lucerne (Medicago sativa L.), was performed, from which 338 unigenes associated with root growth and drought response were identified. Among these unigenes, 27 transcription factors were deduced by functional-domain prediction. The transcription levels of a GRAS and a WRKY transcription factor were confirmed to be responding sensitively to water stresses. Additionally, protein–protein interaction (PPI) networks were predicted for all drought-induced root-related unigenes with different stringency levels. A mitogen-activated protein kinase (MAPK4) and an EF-hand calcium-binding protein (CML24) module were pinpointed to be putative ‘master’ signalling hubs in these PPI networks. Detailed examination of these genomic resources would further reveal key regulators of Lablab drought tolerance.

Molecular and genetic studies of the basis of virulence/avirulence in Meloidogyne chitwoodi

Within the EU project DREAM there is a programme of work investigating the molecular basis of virulence and avirulence in Meloidogyne chitwoodi and which involves partners from the Netherlands, France and Scotland. In Wageningen AFLPs are being used to examine genetic diversity between populations of M. chitwoodi. Partners in Wageningen and INRA are using M. javanica and M. incognita as model systems to isolate genes producing secreted proteins that could be implicated in the host pathogen interaction. They will search for homologous sequences in M. chitwoodi. At Rennes two-dimensional electrophoresis (2DE) studies are being conducted. The PCR based suppressive subtractive hybridisation (SSH), has been used at PRI and SCRI to compare avirulent and virulent nematodes and infected plant material (resistant and susceptible) at different times after infection.

Comparison of gene expression in Solanum bulbocastanum infected with virulent and avirulent isolates of Meloidogyne chitwoodi

Resistance to root knot nematode M. chitwoodi has been identified in the wild tuber-bearing Solanum species, S. bulbocastanum. Three pathotypes were identified suggesting at least two different genetic factors for virulence and resistance in the pathogen and the host species, respectively. Roots of S. bulbocastanum were infested with two isolates of M. chitwoodi differing in virulence. The infection process was monitored by histological examination of roots allowing time points to be identified. cDNA libraries were constructed from infected root tissue using Suppression Subtractive Hybridisation (SSH) to enrich for transcripts from either compatible or incompatible interactions, at three days and seven days post infection. Both plant and nematode genes, which may be important during the host/parasite interaction, were identified.

In vivo-derived horse blastocysts show transcriptional upregulation of developmentally important genes compared with in vitro-produced horse blastocysts

In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 and ODC) were selected to confirm this differential expression by reverse transcription–quantitative real-time polymerase chain reaction (RT-qPCR). Using RT-qPCR, five genes were confirmed to be significantly upregulated in in vivo-derived blastocysts (i.e. FABP3, HSP90AA1 (both P < 0.05), ODC, MOBKL3 and BEX2 (P < 0.005 for all three)), confirming the results of the SSH. There was no significant difference in MCM7 expression between IVP and in vivo-derived blastocysts. In conclusion, five genes that are transcriptionally upregulated in in vivo-derived equine blastocysts compared with IVP blastocysts have been identified. Because of their possible importance in embryonic development, the expression of these genes can be used as a marker to evaluate in vitro embryo production systems in the horse.