Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid.

The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-like 9 (NTL9) and CCA1 hiking expedition (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, phytoalexin-deficient 4 (PAD4) and enhanced disease susceptibility 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators calmodulin binding protein 60g (CBP60g) and systemic acquired resistance deficient 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production.

Biological inquiry: a new course and assessment plan in response to the call to transform undergraduate biology.

We transformed our first-year curriculum in biology with a new course, Biological Inquiry, in which >50% of all incoming, first-year students enroll. The course replaced a traditional, content-driven course that relied on outdated approaches to teaching and learning. We diversified pedagogical practices by adopting guided inquiry in class and in labs, which are devoted to building authentic research skills through open-ended experiments. Students develop core biological knowledge, from the ecosystem to molecular level, and core skills through regular practice in hypothesis testing, reading primary literature, analyzing data, interpreting results, writing in disciplinary style, and working in teams. Assignments and exams require higher-order cognitive processes, and students build new knowledge and skills through investigation of real-world problems (e.g., malaria), which engages students' interest. Evidence from direct and indirect assessment has guided continuous course revision and has revealed that compared with the course it replaced, Biological Inquiry produces significant learning gains in all targeted areas. It also retains 94% of students (both BA and BS track) compared with 79% in the majors-only course it replaced. The project has had broad impact across the entire college and reflects the input of numerous constituencies and close collaboration among biology professors and students.

Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation.

Phytopathogens can manipulate plant hormone signaling to access nutrients and counteract defense responses. Pseudomonas syringae produces coronatine, a toxin that mimics the plant hormone jasmonic acid isoleucine and promotes opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility, and disease symptoms. We examined the mechanisms underlying coronatine-mediated virulence and show that coronatine activates three homologous NAC transcription factor (TF) genes, ANAC019, ANAC055, and ANAC072, through direct activity of the TF, MYC2. Genetic characterization of NAC TF mutants demonstrates that these TFs mediate coronatine-induced stomatal reopening and bacterial propagation in both local and systemic tissues by inhibiting the accumulation of the key plant immune signal salicylic acid (SA). These NAC TFs exert this inhibitory effect by repressing ICS1 and activating BSMT1, genes involved in SA biosynthesis and metabolism, respectively. Thus, a signaling cascade by which coronatine confers its multiple virulence activities has been elucidated.

Arabidopsis BRCA2 and RAD51 proteins are specifically involved in defense gene transcription during plant immune responses.

Systemic acquired resistance (SAR) is a plant immune response associated with both transcriptional reprogramming and increased homologous DNA recombination (HR). SNI1 is a negative regulator of SAR and HR, as indicated by the increased basal expression of defense genes and HR in sni1. We found that the sni1 phenotypes are rescued by mutations in BREAST CANCER 2 (BRCA2). In humans, BRCA2 is a mediator of RAD51 in pairing of homologous DNA. Mutations in BRCA2 cause predisposition to breast/ovarian cancers; however, the role of the BRCA2-RAD51 complex in transcriptional regulation remains unclear. In Arabidopsis, both brca2 and rad51 were found to be hypersusceptible not only to genotoxic substances, but also to pathogen infections. A whole-genome microarray analysis showed that downstream of NPR1, BRCA2A is a major regulator of defense-related gene transcription. ChIP demonstrated that RAD51 is specifically recruited to the promoters of defense genes during SAR. This recruitment is dependent on the SAR signal salicylic acid (SA) and on the function of BRCA2. This study provides the molecular evidence showing that the BRCA2-RAD51 complex, known for its function in HR, also plays a direct and specific role in transcription regulation during plant immune responses.

Inhibition of superoxide generation upon T-cell receptor engagement rescues Mart-1(27-35)-reactive T cells from activation-induced cell death.

Cytotoxic T lymphocytes (CTL) may undergo massive expansion upon appropriate antigenic stimulation. Homeostasis is maintained by a subsequent "contraction" of these cells. Activation-induced cell death (AICD) and programmed cell death prevent the untoward side effects, arising from excessive numbers and prolonged persistence of activated CTL, that occur upon uncontrolled and/or continued expansion. However, effector cell persistence has been identified as a hallmark of successful T-cell-mediated adoptive immunotherapy. Thus, prevention of AICD may be critical to achieve more successful clinical results. We have previously shown that treatment with the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 protects human melanoma epitope Mart-1(27-35)-reactive CTL from apoptotic death upon their reencounter with cognate antigen. However, inhibition of JNK also interferes with the functional ability of the CTL to secrete IFN-gamma. Here, we show that reactive oxygen species (ROS) inhibitors, such as the superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), efficiently protected Mart-1(27-35)-reactive primary CTL from AICD without impairing their functional capability. MnTBAP prevented the increase in intracellular ROS, mitochondrial membrane collapse, and DNA fragmentation observed in control-treated cells upon cognate antigen encounter. Furthermore, the mechanism of AICD prevention in primary CTL included blockade of JNK activation. Finally, tumor-reactive in vitro expanded tumor infiltrating lymphocytes, which are used clinically in cancer immunotherapy, also benefit from MnTBAP-mediated antioxidant treatment. Thus, modulation of the redox pathway might improve CTL persistence and lead to better clinical results for T cell-based immunotherapies.

Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity.

Systemic acquired resistance (SAR) is a broad-spectrum plant immune response involving profound transcriptional changes that are regulated by the coactivator NPR1. Nuclear translocation of NPR1 is a critical regulatory step, but how the protein is regulated in the nucleus is unknown. Here, we show that turnover of nuclear NPR1 protein plays an important role in modulating transcription of its target genes. In the absence of pathogen challenge, NPR1 is continuously cleared from the nucleus by the proteasome, which restricts its coactivator activity to prevent untimely activation of SAR. Surprisingly, inducers of SAR promote NPR1 phosphorylation at residues Ser11/Ser15, and then facilitate its recruitment to a Cullin3-based ubiquitin ligase. Turnover of phosphorylated NPR1 is required for full induction of target genes and establishment of SAR. These in vivo data demonstrate dual roles for coactivator turnover in both preventing and stimulating gene transcription to regulate plant immunity.

A high-performance, small-scale microarray for expression profiling of many samples in Arabidopsis-pathogen studies.

Studies of the behavior of biological systems often require monitoring of the expression of many genes in a large number of samples. While whole-genome arrays provide high-quality gene-expression profiles, their high cost generally limits the number of samples that can be studied. Although inexpensive small-scale arrays representing genes of interest could be used for many applications, it is challenging to obtain accurate measurements with conventional small-scale microarrays. We have developed a small-scale microarray system that yields highly accurate and reproducible expression measurements. This was achieved by implementing a stable gene-based quantile normalization method for array-to-array normalization, and a probe-printing design that allows use of a statistical model to correct for effects of print tips and uneven hybridization. The array measures expression values in a single sample, rather than ratios between two samples. This allows accurate comparisons among many samples. The array typically yielded correlation coefficients higher than 0.99 between technically duplicated samples. Accuracy was demonstrated by a correlation coefficient of 0.88 between expression ratios determined from this array and an Affymetrix GeneChip, by quantitative RT-PCR, and by spiking known amounts of specific RNAs into the RNA samples used for profiling. The array was used to compare the responses of wild-type, rps2 and ndr1 mutant plants to infection by a Pseudomonas syringae strain expressing avrRpt2. The results suggest that ndr1 affects a defense-signaling pathway(s) in addition to the RPS2-dependent pathway, and indicate that the microarray is a powerful tool for systems analyses of the Arabidopsis disease-signaling network.