Arabidopsis GOLDEN2-LIKE (GLK) transcription factors activate jasmonic acid (JA)-dependent disease susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis, as well as JA-independent plant immunity against the necrotrophic pathogen Botrytis cinerea.

Arabidopsis thaliana GOLDEN2-LIKE (GLK1 and 2) transcription factors regulate chloroplast development in a redundant manner. Overexpression of AtGLK1 (35S:AtGLK1) in Arabidopsis also confers resistance to the cereal pathogen Fusarium graminearum. To further elucidate the role of GLK transcription factors in plant defence, the Arabidopsis glk1 glk2 double-mutant and 35S:AtGLK1 plants were challenged with the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2. Compared with Col-0, glk1 glk2 plants were highly resistant to Hpa Noco2, whereas 35S:AtGLK1 plants showed enhanced susceptibility to this pathogen. Genetic studies suggested that AtGLK-mediated plant defence to Hpa Noco2 was partially dependent on salicylic acid (SA) accumulation, but independent of the SA signalling protein NONEXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1). Pretreatment with jasmonic acid (JA) dramatically reversed Hpa Noco2 resistance in the glk1 glk2 double mutant, but only marginally affected the 35S:AtGLK1 plants. In addition, overexpression of AtGLK1 in the JA signalling mutant coi1-16 did not increase susceptibility to Hpa Noco2. Together, our GLK gain-of-function and loss-of-function experiments suggest that GLK acts upstream of JA signalling in disease susceptibility to Hpa Noco2. In contrast, glk1 glk2 plants were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, whereas 35S:AtGLK1 plants exhibited heightened resistance which could be maintained in the absence of JA signalling. Together, the data reveal that AtGLK1 is involved in JA-dependent susceptibility to the biotrophic pathogen Hpa Noco2 and in JA-independent resistance to the necrotrophic pathogen B. cinerea.

Found in translation: high-throughput chemical screening in Arabidopsis thaliana identifies small molecules that reduce Fusarium head blight disease in wheat.

Despite the tremendous economic impact of cereal crop pathogens such as the fungus Fusarium graminearum, the development of strategies for enhanced crop protection is hampered by complex host genetics and difficulties in performing high-throughput analyses. To bypass these challenges, we have developed an assay in which the interaction between F. graminearum and the model plant Arabidopsis thaliana is monitored in liquid media in 96-well plates. In this assay, fungal infection is associated with the development of dark lesion-like spots on the cotyledons of Arabidopsis seedlings by 4 days postinoculation. These symptoms can be alleviated by the application of known defense-activating small molecules and in previously described resistant host genetic backgrounds. Based on this infection phenotype, we conducted a small-scale chemical screen to identify small molecules that protect Arabidopsis seedlings from infection by F. graminearum. We identified sulfamethoxazole and the indole alkaloid gramine as compounds with strong protective activity in the liquid assay. Remarkably, these two chemicals also significantly reduced the severity of F. graminearum infection in wheat. As such, the Arabidopsis-based liquid assay represents a biologically relevant surrogate system for high-throughput studies of agriculturally important plant-pathogen interactions.

The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli.

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins. Two Brassica napus Group 3 LEA proteins, BN115m and a truncated fragment of BNECP63, were fused to two target proteins identified as recalcitrant to overexpression in soluble form or outside of inclusion bodies. Fusion of a truncated peptide of BNECP63 is sufficient to provide soluble and high levels of recombinant overexpression of BNPsbS (an intrinsic membrane chlorophyll-binding protein of photosystem II light harvesting complex) and a peptide of the Hepatitis C viral polyprotein. Furthermore, fusion of the recombinant target proteins to BNECP63 or BN115 prevented irreversible heat- and freeze-induced precipitation. These experiments not only underscore the exploitation of LEA-type peptides in facilitating protein overexpression and protection, but also provide insights into the mechanism of LEA proteins in cellular protection.

The GLK1 'regulon' encodes disease defense related proteins and confers resistance to Fusarium graminearum in Arabidopsis.

Overexpression (OE) was used to study the role of the Arabidopsis Golden2-like (GLK1) transcriptional activator in regulating gene expression. Affymetrix Gene Chip and RT-PCR analyses indicated that GLK1 OE in Arabidopsis reprogrammed gene expression networks to enhance a high constitutive expression of genes encoding disease defense related proteins. These include PR10, isochorismate synthase, antimicrobial peptides, glycosyl hydrolases, MATE efflux and other genes associated with pathogen response and detoxification. However, PR1, an indicator of systemic acquired resistance (SAR), was downregulated in GLK1 OE. GLK1 OE in Arabidopsis confers resistance to Fusarium graminearum, a broad host pathogen responsible for major losses in cereal crops. This is the first identification of the GLK1 'regulon' and a novel role for GLK1 in plant defense, suggesting its potential use for providing disease resistance in crop plants.

The effect of overexpression of two Brassica CBF/DREB1-like transcription factors on photosynthetic capacity and freezing tolerance in Brassica napus.

The effects of overexpression of two Brassica CBF/DREB1-like transcription factors (BNCBF5 and 17) in Brassica napus cv. Westar were studied. In addition to developing constitutive freezing tolerance and constitutively accumulating COR gene mRNAs, BNCBF5- and 17-overexpressing plants also accumulate moderate transcript levels of genes involved in photosynthesis and chloroplast development as identified by microarray and Northern analyses. These include GLK1- and GLK2-like transcription factors involved in chloroplast photosynthetic development, chloroplast stroma cyclophilin ROC4 (AtCYP20-3), beta-amylase and triose-P/Pi translocator. In parallel with these changes, increases in photosynthetic efficiency and capacity, pigment pool sizes, increased capacities of the Calvin cycle enzymes, and enzymes of starch and sucrose biosynthesis, as well as glycolysis and oxaloacetate/malate exchange are seen, suggesting that BNCBF overexpression has partially mimicked cold-induced photosynthetic acclimation constitutively. Taken together, these results suggest that BNCBF/DREB1 overexpression in Brassica not only resulted in increased constitutive freezing tolerance but also partially regulated chloroplast development to increase photochemical efficiency and photosynthetic capacity.

Regulation and characterization of four CBF transcription factors from Brassica napus.

Four orthologues of the Arabidopsis CBF/Dreb transcriptional activator genes were isolated from the winter Brassica napus, cv. Jet neuf. All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences suggested that BNCBFs 5, 7and 16 are very similar to the Arabidopsis CBFI whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain. Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the Brassica seedlings and preceded transcript accumulation of the cold-inducible BN28 gene, a Brassica orthologue of the cor6.6 or KIN gene from Arabidopsis. Cold-induced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16. Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress. BNCBF17 was not responsive to salt stress. BNCBF transcript accumulation was similar in both spring and winter Brassica but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type. BNCBF5 and 17 proteins bind in vitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters. Differential binding preferences, however, to LTREs between BNI 15 and BN28 were observed. Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5. Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17 trans-activation of the Lac-Z reporter gene in yeast. We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in Brassica, controlling the expression of cold-induced genes through an ABA-independent pathway.