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Accumulating evidence suggests that chloroplasts are an important battleground during various microbe-host interactions. Plants have evolved layered strategies to reprogram chloroplasts to promote de novo biosynthesis of defense-related phytohormones and the accumulation of reactive oxygen species (ROS). In this minireview, we will discuss how the host controls chloroplast ROS accumulation during effector-triggered immunity (ETI) at the level of selective mRNA decay, translational regulation, and autophagy-dependent formation of Rubisco-containing bodies (RCBs). We hypothesize that regulation at the level of cytoplasmic mRNA decay impairs the repair cycle of photosystem II (PSII) and thus facilitates ROS generation at PSII. Meanwhile, removing Rubisco from chloroplasts potentially reduces both O 2 and NADPH consumption. As a consequence, an over-reduced stroma would further exacerbate PSII excitation pressure and enhance ROS production at photosystem I.

Plants utilize an innate immune system to protect themselves from disease. While many molecular components of plant innate immunity resemble the innate immunity of animals, plants also have evolved a number of truly unique defense mechanisms, particularly at the physiological level. Plant's flexible developmental program allows them the unique ability to simply produce new organs as needed, affording them the ability to replace damaged organs. Here we develop a system to study pathogen-triggered leaf abscission in Arabidopsis. Cauline leaves infected with the bacterial pathogen Pseudomonas syringae abscise as part of the defense mechanism. Pseudomonas syringae lacking a functional type III secretion system fail to elicit an abscission response, suggesting that the abscission response is a novel form of immunity triggered by effectors. HAESA/HAESA-like 2, INFLORESCENCE DEFICIENT IN ABSCISSION, and NEVERSHED are all required for pathogen-triggered abscission to occur. Additionally phytoalexin deficient 4, enhanced disease susceptibility 1, salicylic acid induction-deficient 2, and senescence-associated gene 101 plants with mutations in genes necessary for bacterial defense and salicylic acid signaling, and NahG transgenic plants with low levels of salicylic acid fail to abscise cauline leaves normally. Bacteria that physically contact abscission zones trigger a strong abscission response; however, long-distance signals are also sent from distal infected tissue to the abscission zone, alerting the abscission zone of looming danger. We propose a threshold model regulating cauline leaf defense where minor infections are handled by limiting bacterial growth, but when an infection is deemed out of control, cauline leaves are shed. Together with previous results, our findings suggest that salicylic acid may regulate both pathogen- and drought-triggered leaf abscission.

Phytopathogenic microbes use secreted effector proteins to increase their virulence in planta . If these effectors or the results of their activity are detected by the plant cell, the plant will mount an immune response which applies evolutionary pressure by reducing growth and success of the pathogen. Bacterial effector proteins in the AvrRps4 family (AvrRps4, HopK1, and XopO) have commonly been used as tools to investigate plant immune components. At the same time, the in planta functions of this family of effectors have yet to be fully characterized. In this minireview we summarize current knowledge about the AvrRps4 effector family with emphasis on properties of the proteins themselves. We hypothesize that the HopK1 C-terminus and the AvrRps4 C-terminus, though unrelated in sequence and structure, are broadly related in functions that counteract plant defense responses.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

In , TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factors (TF) play critical functions in developmental processes. Recent studies suggest they also function in plant immunity, but whether they play an important role in systemic acquired resistance (SAR) is still unknown. NON-EXPRESSER OF PR GENES 1 (NPR1), as an essential transcriptional regulatory node in SAR, exerts its regulatory role in downstream genes expression through interaction with TFs. In this work, we provide biochemical and genetic evidence that TCP8, TCP14, and TCP15 are involved in the SAR signaling pathway. TCP8, TCP14, and TCP15 physically interacted with NPR1 in yeast two-hybrid assays, and these interactions were further confirmed . SAR against the infection of virulent strain pv. ( ) ES4326 in the triple T-DNA insertion mutant was partially compromised compared with Columbia 0 (Col-0) wild type plants. The induction of SAR marker genes , and in local and systemic leaves was dramatically decreased in the mutant compared with that in Col-0 after local treatment with ES4326 carrying . Results from yeast one-hybrid and chromatin immunoprecipitation (ChIP) assays demonstrated that TCP15 can bind to a conserved TCP binding motif, GCGGGAC, within the promoter of , and this binding was enhanced by NPR1. Results from RT-qPCR assays showed that TCP15 promotes the expression of in response to salicylic acid induction. Taken together, these data reveal that TCP8, TCP14, and TCP15 physically interact with NPR1 and function redundantly to establish SAR, that TCP15 promotes the expression of through directly binding a TCP binding site within the promoter of , and that this binding is enhanced by NPR1.

The Arabidopsis (Arabidopsis thaliana) disease resistance protein RESISTANCE TO PSEUDOMONAS SYRINGAE4 (RPS4) activates defenses in response to bacterial pathogens expressing avrRps4 in a gene-for-gene specific manner. The RPS4 gene produces multiple transcripts via alternative splicing of two regular introns flanking exon 3 and a cryptic intron within exon 3. We showed previously that RPS4-mediated resistance requires the combined presence of transcripts encoding both full-length and truncated open reading frames. Here, we demonstrate that alternative splicing of RPS4 undergoes dynamic changes specifically during the resistance response. Furthermore, RPS4 expression was induced by the presence of AvrRps4 in an EDS1-dependent manner. Interestingly, inducible alternative splicing was not limited to the avrRps4-RPS4 interaction, indicating that regulation of alternative splicing may be a general response to prime the plant stress response system. Intron-deficient transgenes lacking only one intron were previously shown to be nonfunctional. Here, we establish quantitatively that the absence of one intron had no effect on the splicing frequency of remaining introns. Given the lack of functionality of single intron-deficient transgenes, this suggests that the products of individual transcripts have distinct functions during RPS4-triggered resistance. Transient expression of truncated RPS4 proteins in Nicotiana benthamiana induced hypersensitive response-like cell death in the absence of AvrRps4. Interestingly, different truncated proteins had markedly differing stability. In summary, RPS4 function is regulated at multiple levels, including gene expression, alternative splicing, and protein stability, presumably to fine-tune activity and limit damage inflicted by activated RPS4 protein.

We report the results of a genome-wide analysis of transcription in Arabidopsis thaliana after treatment with Pseudomonas syringae pathovar tomato. Our time course RNA-Seq experiment uses over 500 million read pairs to provide a detailed characterization of the response to infection in both susceptible and resistant hosts. The set of observed differentially expressed genes is consistent with previous studies, confirming and extending existing findings about genes likely to play an important role in the defense response to Pseudomonas syringae. The high coverage of the Arabidopsis transcriptome resulted in the discovery of a surprisingly large number of alternative splicing (AS) events--more than 44% of multi-exon genes showed evidence for novel AS in at least one of the probed conditions. This demonstrates that the Arabidopsis transcriptome annotation is still highly incomplete, and that AS events are more abundant than expected. To further refine our predictions, we identified genes with statistically significant changes in the ratios of alternative isoforms between treatments. This set includes several genes previously known to be alternatively spliced or expressed during the defense response, and it may serve as a pool of candidate genes for regulated alternative splicing with possible biological relevance for the defense response against invasive pathogens.

Regulation of the plant immune system is important for controlling the specificity and amplitude of responses to pathogens and in preventing growth-inhibiting autoimmunity that leads to reductions in plant fitness. In previous work, we reported that SRFR1, a negative regulator of effector-triggered immunity, interacts with SNC1 and EDS1. When SRFR1 is non-functional in the Arabidopsis accession Col-0, SNC1 levels increase, causing a cascade of events that lead to autoimmunity phenotypes. Previous work showed that some members of the transcriptional co-repressor family TOPLESS interact with SNC1 to repress negative regulators of immunity. Therefore, to explore potential connections between SRFR1 and TOPLESS family members, we took a genetic approach that examined the effect of each TOPLESS member in the srfr1 mutant background. The data indicated that an additive genetic interaction exists between SRFR1 and two members of the TOPLESS family, TPR2 and TPR3 , as demonstrated by increased stunting and elevated PR2 expression in srfr1 tpr2 and srfr1 tpr2 tpr3 mutants. Furthermore, the tpr2 mutation intensifies autoimmunity in the auto-active snc1-1 mutant, indicating a novel role of these TOPLESS family members in negatively regulating SNC1 -dependent phenotypes. This negative regulation can also be reversed by overexpressing TPR2 in the srfr1 tpr2 background. Similar to TPR1 that positively regulates snc1-1 phenotypes by interacting with SNC1, we show here that TPR2 directly binds the N-terminal domain of SNC1. In addition, TPR2 interacts with TPR1 in vivo , suggesting that the opposite functions of TPR2 and TPR1 are based on titration of SNC1-TPR1 complexes by TPR2 or altered functions of a SNC1-TPR1-TPR2 complex. Thus, this work uncovers diverse functions of individual members of the TOPLESS family in Arabidopsis and provides evidence for the additive effect of transcriptional and post-transcriptional regulation of SNC1 .

The Arabidopsis thaliana AtOPT3 belongs to the oligopeptide transporter (OPT) family, a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi, and plants. A null mutation in AtOPT3 resulted in embryo lethality, indicating an essential role for AtOPT3 in embryo development. In this article, we report on the isolation and phenotypic characterization of a second AtOPT3 mutant line, opt3-2, harboring a T-DNA insertion in the 5' untranslated region of AtOPT3. The T-DNA insertion in the AtOPT3 promoter resulted in reduced but sufficient AtOPT3 expression to allow embryo formation in opt3-2 homozygous seeds. Phenotypic analyses of opt3-2 plants revealed three interesting loss-of-function phenotypes associated with iron metabolism. First, reduced AtOPT3 expression in opt3-2 plants resulted in the constitutive expression of root iron deficiency responses regardless of exogenous iron supply. Second, deregulation of root iron uptake processes in opt3-2 roots resulted in the accumulation of very high levels of iron in opt3-2 tissues. Hyperaccumulation of iron in opt3-2 resulted in the formation of brown necrotic areas in opt3-2 leaves and was more pronounced during the seed-filling stage. Third, reduced AtOPT3 expression resulted in decreased accumulation of iron in opt3-2 seeds. The reduced accumulation of iron in opt3-2 seeds is especially noteworthy considering the excessively high levels of accumulated iron in other opt3-2 tissues. AtOPT3, therefore, plays a critical role in two important aspects of iron metabolism, namely, maintenance of whole-plant iron homeostasis and iron nutrition of developing seeds.

The Pseudomonas syringae-Arabidopsis (Arabidopsis thaliana) interaction is an extensively studied plant-pathogen system. Arabidopsis possesses approximately 150 putative resistance genes encoding nucleotide binding site (NBS) and leucine-rich repeat (LRR) domain-containing proteins. The majority of these belong to the Toll/Interleukin-1 receptor (TIR)-NBS-LRR (TNL) class. Comparative studies with the coiled-coil-NBS-LRR genes RPS2, RPM1, and RPS5 and isogenic P. syringae strains expressing single corresponding avirulence genes have been particularly fruitful in dissecting specific and common resistance signaling components. However, the major TNL class is represented by a single known P. syringae resistance gene, RPS4. We previously identified hopA1 from P. syringae pv syringae strain 61 as an avirulence gene that signals through ENHANCED DISEASE SUSCEPTIBILITY1, indicating that the corresponding resistance gene RPS6 belongs to the TNL class. Here we report the identification of RPS6 based on a forward-genetic screen and map-based cloning. Among resistance proteins of known function, the deduced amino acid sequence of RPS6 shows highest similarity to the TNL resistance protein RAC1 that determines resistance to the oomycete pathogen Albugo candida. Similar to RPS4 and other TNL genes, RPS6 generates alternatively spliced transcripts, although the alternative transcript structures are RPS6 specific. We previously characterized SRFR1 as a negative regulator of avrRps4-triggered immunity. Interestingly, mutations in SRFR1 also enhanced HopA1-triggered immunity in rps6 mutants. In conclusion, the cloning of RPS6 and comparisons with RPS4 will contribute to a closer dissection of the TNL resistance pathway in Arabidopsis.