ML3: a novel regulator of herbivory-induced responses in Arabidopsis thaliana.

ML (MD2-related lipid recognition) proteins are known to enhance innate immune responses in mammals. This study reports the analysis of the putative ML gene family in Arabidopsis thaliana and suggests a role for the ML3 gene in herbivory-associated responses in plants. Feeding by larvae of the Lepidopteran generalist herbivore Spodoptera littoralis and larvae of the specialist herbivore Plutella xylostella activated ML3 transcription in leaf tissues. ML3 loss-of-function Arabidopsis plants were compromised in the upregulation of herbivory-induced genes and displayed a semi-dwarf phenotype. Herbivory bioassays showed that larvae of S. littoralis fed on ml3 mutant plants gained more weight compared to larvae fed on wild-type plants while larvae of P. xylostella did not show any significant difference. Virus-induced gene silencing of ML3 expression in plants compromised in jasmonic acid (JA) and salicylic acid (SA) signalling revealed a complex role of ML3 in JA/defence signalling affecting both JA- and SA-dependent responses. The data suggest that ML3 is involved in herbivory-mediated responses in Arabidopsis and that it has a potential role in herbivory-associated molecular pattern recognition.

Varied response of Spodoptera littoralis against Arabidopsis thaliana with metabolically engineered glucosinolate profiles.

Upon herbivory glucosinolates are known to be degraded into a cascade of secondary products that can be detrimental for certain herbivores. We performed herbivory bioassays using first and second instar generalist Lepidoptera larvae Spodoptera littoralis on Arabidopsis thaliana engineered to overexpress novel glucosinolates. A differential response in larval feeding patterns was observed on the plants engineered with novel glucosinolates. Larvae fed on plants overexpressing 4-hydroxybenzyl glucosinolate and isopropyl glucosinolate showed little response. Larvae fed on 35S:CYP79A2 plants engineered to overexpress benzyl glucosinolates, however, showed reduced larval and pupal weights. Upon herbivory a high expression of JA signalling gene LOX2 was observed on the 35S:CYP79A2 plants compared to the PR1a and VSP2 expression. To confirm the role of benzyl isothiocyanate (BITC), a degradation product of benzyl glucosinolate overexpressing plants, in the retarded larval growth we used Virus Induced Gene Silencing (VIGS) approach to silence LOX2 expression in the 35S:CYP79A2 plants. S. littoralis larvae fed on LOX2 silenced 35S:CYP79A2 plants exhibited a retarded larval growth thus indicating that BITC played a pivotal role in anti-herbivory and not only the JA signalling pathway.

Two loci in sorghum with NB-LRR encoding genes confer resistance to Colletotrichum sublineolum.

The aim of this work was to identify plant resistance genes to the sorghum anthracnose fungus Colletotrichum sublineolum. cDNA-AFLP transcript profiling on two contrasting sorghum genotypes inoculated with C. sublineolum generated about 3,000 informative fragments. In a final set of 126 sequenced genes, 15 were identified as biotic stress related. Seven of the plant-derived genes were selected for functional analysis using a Brome mosaic virus-based virus-induced gene silencing (VIGS) system followed by fungal inoculation and quantitative real-time PCR analysis. The candidate set comprised genes encoding resistance proteins (Cs1A, Cs2A), a lipid transfer protein (SbLTP1), a zinc finger-like transcription factor (SbZnTF1), a rice defensin-like homolog (SbDEFL1), a cell death related protein (SbCDL1), and an unknown gene harboring a casein kinase 2-like domain (SbCK2). Our results demonstrate that down-regulation of Cs1A, Cs2A, SbLTP1, SbZnF1 and SbCD1 via VIGS, significantly compromised the resistance response while milder effects were observed with SbDEFL1 and SbCK2. Expanded genome analysis revealed that Cs1A and Cs2A genes are located in two different loci on chromosome 9 closely linked with duplicated genes Cs1B and Cs2B, respectively. The nucleotide binding-leucine rich repeat (NB-LRR) encoding Cs gene sequence information is presently employed in regional breeding programs.

A highly conserved NB-LRR encoding gene cluster effective against Setosphaeria turcica in sorghum.

BACKGROUND: The fungal pathogen Setosphaeria turcica causes turcicum or northern leaf blight disease on maize, sorghum and related grasses. A prevalent foliar disease found worldwide where the two host crops, maize and sorghum are grown. The aim of the present study was to find genes controlling the host defense response to this devastating plant pathogen. A cDNA-AFLP approach was taken to identify candidate sequences, which functions were further validated via virus induced gene silencing (VIGS), and real-time PCR analysis. Phylogenetic analysis was performed to address evolutionary events. RESULTS: cDNA-AFLP analysis was run on susceptible and resistant sorghum and maize genotypes to identify resistance-related sequences. One CC-NB-LRR encoding gene GRMZM2G005347 was found among the up-regulated maize transcripts after fungal challenge. The new plant resistance gene was designated as St referring to S. turcica. Genome sequence comparison revealed that the CC-NB-LRR encoding St genes are located on chromosome 2 in maize, and on chromosome 5 in sorghum. The six St sorghum genes reside in three pairs in one locus. When the sorghum St genes were silenced via VIGS, the resistance was clearly compromised, an observation that was supported by real-time PCR. Database searches and phylogenetic analysis suggest that the St genes have a common ancestor present before the grass subfamily split 50-70 million years ago. Today, 6 genes are present in sorghum, 9 in rice and foxtail millet, respectively, 3 in maize and 4 in Brachypodium distachyon. The St gene homologs have all highly conserved sequences, and commonly reside as gene pairs in the grass genomes. CONCLUSIONS: Resistance genes to S. turcica, with a CC-NB-LRR protein domain architecture, have been found in maize and sorghum. VIGS analysis revealed their importance in the surveillance to S. turcica in sorghum. The St genes are highly conserved in sorghum, rice, foxtail millet, maize and Brachypodium, suggesting an essential evolutionary function.

Enhancer trapping identifies TRI, an Arabidopsis gene up-regulated by pathogen infection.

Enhancer trap Arabidopsis thaliana plants were screened for genes up-regulated by virus infection. The plants carried T-DNA insertions comprising a minimal -60-bp Cauliflower mosaic virus 35S promoter fused to the beta-glucuronidase (GUS) reporter gene. Approximately 12,000 plants were assayed for GUS activity before and after rub-inoculation with Tobacco rattle virus (TRV) tagged with the green fluorescent protein (GFP). One plant and its progeny consistently showed upregulation of GUS activity in response to TRV-GFP infection, indicating that a virus-responsive enhancer element was "tagged" by the T-DNA in this line. Other viruses, bacteria, and oomycetes, but not wounding, up-regulated GUS activity in the enhancer trap line, indicating that the response was not specific to TRV-GFP infection. A pathogen-inducible, alternatively spliced gene was identified, which we have termed TRI for TRV-induced gene. A pathogen-responsive element was localized to a 1.1-kb region upstream of the T-DNA insertion, and two different cis-acting elements, both implicated in defense responses, were found in the sequence upstream of TRI. Sequence analyses revealed that TRI is similar to ACRE169, a gene that is up-regulated in Cf-9-expressing tobacco when treated with Avr-9, the Cladosporium fulvum elicitor of the Cf-9 resistance response.

TIP, a novel host factor linking callose degradation with the cell-to-cell movement of Potato virus X.

The cell-to-cell movement of Potato virus X (PVX) requires four virus-encoded proteins, the triple gene block (TGB) proteins (TGB25K, TGB12K, and TGB8K) and the coat protein. TGB12K increases the plasmodesmal size exclusion limit (SEL) and may, therefore, interact directly with components of the cell wall or with plant proteins associated with bringing about this change. A yeast two-hybrid screen using TGB12K as bait identified three TGB12K-interacting proteins (TIP1, TIP2, and TIP3). All three TIPs interacted specifically with TGB12K but not with TGB25K or TGB8K. Similarly, all three TIPs interacted with beta-1,3-glucanase, the enzyme that may regulate plasmodesmal SEL through callose degradation. Sequence analyses revealed that the TIPs encode very similar proteins and that TIP1 corresponds to the tobacco ankyrin repeat-containing protein HBP1. A TIP1::GFP fusion protein localized to the cytoplasm. Coexpression of this fusion protein with TGB12K induced cellular changes manifested as deposits of additional cytoplasm at the cell periphery. This work reports a direct link between a viral movement protein required to increase plasmodesmal SEL and a host factor that has been implicated as a key regulator of plasmodesmal SEL. We propose that the TIPs are susceptibility factors that modulate the plasmodesmal SEL.

STY1 and STY2 promote the formation of apical tissues during Arabidopsis gynoecium development.

Gynoecium ontogenesis in Arabidopsis is accomplished by the co-ordinated activity of genes that control patterning and the regional differentiation of tissues, and ultimately results in the formation of a basal ovary, a short style and an apical stigma. A transposon insertion in the STYLISH1 (STY1) gene results in gynoecia with aberrant style morphology, while an insertion mutation in the closely related STYLISH2 (STY2) gene has no visible effect on gynoecium development. However, sty1-1 sty2-1 double mutant plants exhibit an enhanced sty1-1 mutant phenotype and are characterized by a further reduction in the amount of stylar and stigmatic tissues and decreased proliferation of stylar xylem. These data imply that STY1 and STY2 are partially redundant and that both genes promote style and stigma formation and influence vascular development during Arabidopsis gynoecium development. Consistently, STY1 and STY2 are expressed in the apical parts of the developing gynoecium and ectopic expression of either STY1 or STY2 driven by the CaMV 35S promoter is sufficient to transform valve cells into style cells. STY1::GUS and STY2::GUS activity is detected in many other organs as well as the gynoecium, suggesting that STY1 and STY2 may have additional functions. This is supported by the sty1-1 sty2-1 double mutants producing rosette and cauline leaves with a higher degree of serration than wild-type leaves. STY1 and STY2 are members of a small gene family, and encode proteins with a RING finger-like motif. Double mutant analyses indicate that STY1 genetically interacts with SPATULA and possibly also with CRABS CLAW.