Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco.

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::ß-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

GmRIN4 protein family members function nonredundantly in soybean race-specific resistance against Pseudomonas syringae.

The Pseudomonas syringae effector AvrB interacts with four related soybean (Glycine max) proteins (GmRIN4a-d), three (GmRIN4b, c, d) of which also interact with the cognate resistance (R) protein, Rpg1-b. Here, we investigated the specific requirements for the GmRIN4 proteins in R-mediated resistance and examined the mechanism of Rpg1-b activation. Using virus-induced gene silencing, we show that only GmRIN4a and b are required for Rpg1-b-mediated resistance. In planta binding assays show that GmRIN4a can associate with Rpg1-b indirectly via GmRIN4b. Pathogen-delivered AvrB induces the phosphorylation of GmRIN4b alone, and prevents interactions between GmRIN4b and Rpg1-b or GmRIN4a. Consistent with this result, a phosphomimic derivative of GmRIN4b (pm4b) fails to bind Rpg1-b and GmRIN4a. Conversely, a phosphodeficient derivative of GmRIN4b (pd4b) continues to bind the R protein and GmRIN4a, in the presence of AvrB. Coexpression of Rpg1-b with pm4b, but not GmRIN4b or pd4b, induces cell death and ion leakage in the heterologous Nicotiana benthamiana. Our data suggest that the AvrB-induced phosphorylation of GmRIN4b, and the subsequent inhibition of interaction among GmRIN4b, GmRIN4a and Rpg1-b, might activate the R protein. Furthermore, even though GmRIN4c and d are not required for Rpg1-b-derived resistance, they do function in resistance derived from other R loci.

The N-terminal extension domain of the C. elegans half-molecule ABC transporter, HMT-1, is required for protein-protein interactions and function.

BACKGROUND: Members of the HMT-1 (heavy metal tolerance factor 1) subfamily of the ATP-binding cassette (ABC) transporter superfamily detoxify heavy metals and have unique topology: they are half-molecule ABC transporters that, in addition to a single transmembrane domain (TMD1) and a single nucleotide-binding domain (NBD1), possess a hydrophobic NH2-terminal extension (NTE). These structural features distinguish HMTs from other ABC transporters in different species including Drosophila and humans. Functional ABC transporters, however, are comprised of at least four-domains (two TMDs and two NDBs) formed from either a single polypeptide or by the association of two or four separate subunits. Whether HMTs act as oligomers and what role the NTE domain plays in their function have not been determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the oligomeric status of Caenorhabditis elegans HMT-1 and the functional significance of its NTE using gel-filtration chromatography in combination with the mating-based split-ubiquitin yeast two-hybrid system (mbSUS) and functional in vivo assays. We found that HMT-1 exists in a protein complex in C. elegans. Studies in S. cerevisiae showed that HMT-1 at a minimum homodimerizes and that oligomerization is essential for HMT-1 to confer cadmium tolerance. We also established that the NTE domain plays an important structural and functional role: it is essential for HMT-1 oligomerization and Cd-detoxification function. However, the NTE itself was not sufficient for oligomerization suggesting that multiple structural features of HMT-1 must associate to form a functional transporter. CONCLUSIONS: The prominence of heavy metals as environmental toxins and the remarkable conservation of HMT-1 structural architecture and function in different species reinforce the value of continued studies of HMT-1 in model systems for identifying functional domains in HMT1 of humans.

Antioxidant response of wheat roots to drought acclimation.

Wheat (Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods. The antioxidant defense in terms of redox metabolites and enzymes in root cells and mitochondria was examined in relation to membrane damage. Acclimated seedlings exhibited higher relative water content and were able to limit the accumulation of H(2)O(2) and membrane damage during subsequent severe water stress conditions. This was due to systematic up-regulation of superoxide dismutase, ascorbate peroxidase (APX), catalase, peroxidases, and ascorbate-glutathione cycle components at both the whole cell level as well as in mitochondria. In contrast, direct exposure of severe water stress to non-acclimated seedlings caused greater water loss, excessive accumulation of H(2)O(2) followed by elevated lipid peroxidation due to the poor antioxidant enzyme response particularly of APX, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and ascorbate-glutathione redox balance. Mitochondrial antioxidant defense was found to be better than the cellular defense in non-acclimated roots. Termination of stress followed by rewatering leads to a rapid enhancement in all the antioxidant defense components in non-acclimated roots, which suggested that the excess levels of H(2)O(2) during severe water stress conditions might have inhibited or down-regulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced tolerance toward oxidative stress in the root tissue of wheat seedlings due to both reactive oxygen species restriction and well-coordinated induction of antioxidant defense.