For thousands of years, humans have been improving crops to better suit their needs. These enhancements are driven by changes in the genetic makeup of the plant. While this was initially ...unintentional, there has been a steady push to increase the pace and precision of crop breeding, something that has occurred alongside a growing understanding of genetics and an escalating capacity to thoroughly assess genomes at the molecular level. With the advent and rapid uptake of molecular breeding techniques, such as transgenics and genome editing over the past few decades, there has been much trepidation regarding the possibility of off-target effects derived from unanticipated mutations at loci other than those intended for alteration, and the unintended risks that this might confer. These concerns persist regardless of the fact that a growing number of studies indicate that the occurrence of off-target mutations derived from newer biotechnological breeding techniques are negligible compared to what is observed with many conventional breeding approaches, and even spontaneously from one generation to the next. Given the impending food security crisis that we are facing in the short-term, there is a critical need to implement a wide range of breeding tools as a means of meeting growing demand, withstanding climate change-related pressures, increasing nutrition, and providing environmental benefits. While food safety is clearly of the utmost importance, now is certainly not the time to prevent the use of particular breeding technologies based on unfounded doubts. Therefore, in this review, we attempt to shed light on these apprehensions by putting purported "risks" into the context of plant breeding as a whole by comparing frequencies of spontaneous mutations with those (both anticipated and unanticipated) that occur through various conventional and biotechnological breeding approaches, including transgenics and genome editing. We then consider how these changes may, or may not, translate into unanticipated risk, and discuss the current global regulatory asynchrony surrounding genome edited crops.
The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant ...avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.
Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from ...sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maizeinfecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.
Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and ...seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcription-factor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.
As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant- specific protein involved in organeUar genome stability in mitochondria and plastids. ...Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the rash I phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppres- sion lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochon- dria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrlal genome sta- bility. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH 1. MSH1 deple- tion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.
Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion ...of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.
Proper regulation of nuclear-encoded, organelle-targeted genes is crucial for plastid and mitochondrial function. Among these genes, MutS Homolog 1 (MSH1) is notable for generating an assortment of ...mutant phenotypes with varying degrees of penetrance and pleiotropy. Stronger phenotypes have been connected to stress tolerance and epigenetic changes, and in Arabidopsis T-DNA mutants, two generations of homozygosity with the msh1 insertion are required before severe phenotypes begin to emerge. These observations prompted us to examine how msh1 mutants contrast according to generation and phenotype by profiling their respective transcriptomes and small RNA populations.
Using RNA-seq, we analyze pathways that are associated with MSH1 loss, including abiotic stresses such as cold response, pathogen defense and immune response, salicylic acid, MAPK signaling, and circadian rhythm. Subtle redox and environment-responsive changes also begin in the first generation, in the absence of strong phenotypes. Using small RNA-seq we further identify miRNA changes, and uncover siRNA trends that indicate modifications at the chromatin organization level. In all cases, the magnitude of changes among protein-coding genes, transposable elements, and small RNAs increases according to generation and phenotypic severity.
Loss of MSH1 is sufficient to cause large-scale regulatory changes in pathways that have been individually linked to one another, but rarely described all together within a single mutant background. This study enforces the recognition of organelles as critical integrators of both internal and external cues, and highlights the relationship between organelle and nuclear regulation in fundamental aspects of plant development and stress signaling. Our findings also encourage further investigation into potential connections between organelle state and genome regulation vis-á-vis small RNA feedback.
Breast reconstruction is generally discouraged in women with inflammatory breast cancer (IBC) due to concerns with recurrence and poor long-term survival. We aim to determine contemporary trends and ...predictors of breast reconstruction and its impact on oncologic outcomes among women with IBC. A systematic literature review for all studies published up to 15 September 2022 was conducted via MEDLINE, Embase, and the Cochrane Library. Studies comparing women diagnosed with IBC undergoing a mastectomy with or without breast reconstruction were evaluated. The initial search yielded 225 studies, of which nine retrospective cohort studies, reporting 2781 cases of breast reconstruction in 29,058 women with IBC, were included. In the past two decades, immediate reconstruction rates have doubled. Younger age, higher income (>USD 25,000), private insurance, metropolitan residence, and bilateral mastectomy were associated with immediate reconstruction. No significant difference was found in overall survival, breast cancer-specific survival or recurrence rates between women undergoing versus not undergoing (immediate or delayed) reconstruction. There is a paucity of data on delayed breast reconstruction following IBC. Immediate breast reconstruction may be a consideration for select patients with IBC, although prospective data is needed to clarify its safety.
Background & Aims:
Genomic instability in colon cancers is a consequence of chromosomal instability characterized by aneuploidy or defective DNA mismatch repair (MMR) indicated by microsatellite ...instability (MSI). Given that high-frequency MSI (MSI-H) and diploidy are correlated, we determined whether they are independent prognostic variables.
Methods:
Astler–Coller stage B2 and C colon cancers (N = 528) from patients treated in 5-fluorouracil–based adjuvant therapy trials were analyzed for MSI using 11 microsatellite markers. Immunostaining for hMLH1, hMSH2, and p53 proteins was performed. DNA ploidy was analyzed by flow cytometry. Associations with disease-free and overall survival were determined.
Results:
MSI-H was detected in 95 tumors (18%), and 70 (74%) of these were diploid. Tumors showing MSI-H (hazard ratio, 0.65; 95% confidence interval, 0.44–0.96;
P = .023) or loss of MMR proteins (
P = .024) were associated with better overall survival. Improved disease-free and overall survival were found for diploid versus aneuploid/tetraploid tumors (overall survival: hazard ratio, 0.59; 95% confidence interval, 0.43–0.79;
P = .0003). In the subgroups of MSI-H and microsatellite stable (MSS)/low-frequency MSI (MSI-L) tumors, diploidy was associated with better survival. The prognostic impact of ploidy was similar in stage B2 and C tumors. Ploidy did not predict the benefit of 5-fluorouracil–based treatment. When ploidy, MSI, and MMR proteins were analyzed in the same multivariate model, only ploidy remained significant.
Conclusions:
DNA ploidy and MSI-H status were independent prognostic variables, yet ploidy was the strongest marker. Diploidy was associated with better survival in MSI-H and in MSS/MSI-L patient subgroups.
RNA interference (RNAi) acts through transcriptional and post-transcriptional gene silencing of homologous sequences. With the goal of using RNAi as a tool for studying gene function in the related ...basidiomycete cereal pathogens Ustilago hordei and Ustilago maydis, we developed a general purpose RNAi expression vector. Tandem, inverted fragments of the GUS gene were inserted into this vector flanking an intron and used to transform engineered GUS-expressing haploid cells. Down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei, even though corresponding GUS double-stranded RNA was detected in both species. Similarly, when the endogenous bW mating-type gene was targeted by RNAi, mating was reduced only in U. hordei. Our work demonstrates the feasibility of using RNAi in U. hordei and provides experimental support for the observed lack of RNAi components in the U. maydis genome. We hypothesize that the sharply limited transposon complement in U. maydis is a biological consequence of this absence.
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