The miR159-GAMYB pathway: silencing and function of GAMYB homologues amongst diverse plant species

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression in eukaryotes. In plants, many miRNA families mediate silencing of target genes, which are involved in plant development and plant defence. For my thesis, I have been investigating the miR159-GAMYB pathway, which appears conserved from basal vascular plants to angiosperms. GAMYB transcription factors have been demonstrated to have conserved roles of programmed cell death (PCD) in both the seed aleurone and the anther tapetum in a number of different plant species. However, what the functional role of GAMYB is in vegetative tissues remains unknown. In Arabidopsis, miR159 is critical for proper growth, as its inhibition has a strong negative impact on vegetative growth, due to deregulated GAMYB expression. However, gamyb loss-of-functional mutants display a wild-type phenotype, as their expression is silenced to phenotypically inconsequential levels by miR159 in vegetative tissues. This raises two questions: (1) how is GAMYB so strongly silenced; (2) why is GAMYB strongly and widely transcribed in vegetative tissues for it to be then completely repressed by miR159? These two questions were the focus of my thesis. Firstly, how the Arabidopsis MYB33 and MYB65 are so strongly silenced in the model plant Arabidopsis was investigated. Both genes were predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site, composed of two stem-loop (SL) structures; whereas such SL structures were not predicted to form in other GAMYB-like genes that are targeted less efficiently by miR159 for expression regulation. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing mutations to form an artificial stem-loop structure adjacent to a miRNA-binding site restored strong miR159-mediated silencing. Although how these predicted structures promote miR159-mediated silencing are not determined, we speculate that the stem-loop structures in the vicinity of the miR159 binding site promotes accessibility of the binding site, where if adjacent sequences form strong stem structures, they are less likely to base-pair with binding site nucleotides, maintaining high accessibility of the binding site. Interestingly, the RNA SL structures are predicted to reside in GAMYB-like homologues of numerous angiosperm and gymnosperm plant species, arguing that these structures have been integral in the miR159-GAMYB regulatory relationship over a long period of time. In addition, these structures are not present in the Arabidopsis GAMYB-like homologues that are not transcribed in vegetative tissues, suggesting that selection for such structures only occurs for homologues transcribed in vegetative tissues as to prevent their expression and demarcating them as sensitive targets of miR159. Secondly, to investigate the functional role of the miR159-GAMYB pathway, target MIMIC159 (MIM159) transgenes, which can inhibit endogenous miR159 activity, were expressed in a number of Arabidopsis ecotypes, as well as in tobacco and rice. Inhibiting miR159 in all three plant species resulted in similar phenotypic outcomes, predominantly stunted growth and irregular leaf shape. This implies that the function and expression of the miR159-GAMYB pathway is strongly conserved in distant plant species. This raises several questions: why is GAMYB widely transcribed if its expression is strongly silenced by miR159 throughout the plant to result in little to no phenotypic impact; and why has this been strongly conserved across multiple plant species. When miR159 activity is inhibited in MIM159 tobacco leaves, pathways related to plant defence response are most up-regulated. This included PATHOGENESIS-RELATED PROTEIN (PR) mRNA levels that were 100-1000s fold up-regulated compared to wild type, and correlated with deregulated GAMYB expression. This finding suggests that the miR159-GAMYB pathway is involved in the plant defence response to biotic stress. However, PR expression is not up-regulated in Arabidopsis or rice when miR159 is inhibited, suggesting that despite the conserved nature of the miR159-GAMYB pathway, there are species-specific differences in its function

Use of HRMA Proteins and Their Genes for Broad Range Protection of Plants Against Bacterial, Fungal and Viral Pathogens

The use of an avr gene hrmA to induce systematic acquired resistance in plant cells, plant seeds, plant tissues and plants is disclosed. Also disclosed is the use of low level expression of promoters in combination with the hrmA gene to provide broad-spectrum pathogen resistance in plant cells, plant seeds, plant tissues and plants

Sequence of a putative Vitis vinifera PR-1

Research Note

Arabidopsis TAO1 is a TIR-NB-LRR protein that contributes to disease resistance induced by the Pseudomonas syringae effector AvrB

The type III effector protein encoded by avirulence gene B (AvrB) is delivered into plant cells by pathogenic strains of Pseudomonas syringae. There, it localizes to the plasma membrane and triggers immunity mediated by the Arabidopsis coiled-coil (CC)-nucleotide binding (NB)-leucine-rich repeat (LRR) disease resistance protein RPM1. The sequence unrelated type III effector avirulence protein encoded by avirulence gene Rpm1 (AvrRpm1) also activates RPM1. AvrB contributes to virulence after delivery from P. syringae in leaves of susceptible soybean plants, and AvrRpm1 does the same in Arabidopsis rpm1 plants. Conditional overexpression of AvrB in rpm1 plants results in leaf chlorosis. In a genetic screen for mutants that lack AvrB-dependent chlorosis in an rpm1 background, we isolated TAO1 (target of AvrB operation), which encodes a Toll-IL-1 receptor (TIR)-NB-LRR disease resistance protein. In rpm1 plants, TAO1 function results in the expression of the pathogenesis-related protein 1 (PR-1) gene, suggestive of a defense response. In RPM1 plants, TAO1 contributes to disease resistance in response to Pto (P. syringae pathovars tomato) DC3000(avrB), but not against Pto DC3000(avrRpm1). The tao1–5 mutant allele, a stop mutation in the LRR domain of TAO1, posttranscriptionally suppresses RPM1 accumulation. These data provide evidence of genetically separable disease resistance responses to AvrB and AvrRpm1 in Arabidopsis. AvrB activates both RPM1, a CC-NB-LRR protein, and TAO1, a TIR-NB-LRR protein. These NB-LRR proteins then act additively to generate a full disease resistance response to P. syringae expressing this type III effector

Suppression of Cdc27B expression induces plant defence responses

Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans, is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY- 2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase- promoting complex or cyclosome ( APC/ C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B, in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus:: Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.</p

Transcriptional Silencing Elements and Their Binding Factors

The invention features an isolated gene silencing regulatory element that includes 5\u27 TACNNTAC 3\u27. Vectors, transgenic plants and seeds thereof that include such a gene silencing regulatory element are also disclosed. The invention further provides methods of decreasing the transcription of a DNA sequence in a transgenic plant using the isolated gene silencing regulatory element

The endoplasmic reticulum in plant immunity and cell death

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants

The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands

<p>Abstract</p> <p>Background</p> <p>The major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database.</p> <p>Results</p> <p>Structural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.</p> <p>Conclusion</p> <p>The ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.</p

Genetic Dissection of Disease Resistance to the Blue Mold Pathogen, \u3cem\u3ePeronospora tabacina\u3c/em\u3e, in Tobacco

Tobacco blue mold, caused by the obligately biotrophic oomycete pathogen Peronospora tabacina D.B. Adam, is a major foliar disease that results in significant losses in tobacco-growing areas. Natural resistance to P. tabacina has not been identified in any variety of common tobacco. Complete resistance, conferred by RBM1, was found in N. debneyi and was transferred into cultivated tobacco by crossing. In the present study, we characterized the RBM1-mediated resistance to blue mold in tobacco and show that the hypersensitive response (HR) plays an important role in the host defense reactions. Genetic mapping indicated that the disease resistance gene locus resides on chromosome 7. The genetic markers linked to this gene and the genetic map we generated will not only benefit tobacco breeders for variety improvement but will also facilitate the positional cloning of RBM1 for biologists