Identification of a suitable method of inoculation for reducing background effect in mock-inoculated controls during gene expression studies in Arabidopsis thaliana.

The recent advancement in the field of transcriptome and methylome sequencing helped scientists to analyse the gene expression and epigenetic status of different genes. Several genes and their regulatory pathways have been discovered due to research into plant-microbe interactions. Previous research on plant-Agrobacterium interactions found that the method of inoculation (wounding using a syringe), resulted in altered DNA methylation of the host DNA repair gene promoters. The expression study of host defence genes revealed that the method of inoculation masked the host response to bacteria. It could be possible that these method-induced changes could interfere with various defence regulatory pathways, which otherwise would not be triggered by the bacteria alone. Hence, it would be critical to identify an appropriate method of inoculation that could provide more unambiguous interpretation of studies involving gene expression and regulation in plants under bacterial stress. The expression dynamics of two defence genes, PR1 and NPR1, under various combinations of parameters such as three different methods of inoculation, treatment with five different bacterial re-suspending solutions, and at three different post-inoculation time intervals were examined in the model plant Arabidopsis thaliana. The H2O2 and superoxide (O2-) production due to various inoculation methods and re-suspending solutions on the host was also studied. The flood inoculation method, which used sterile deionized water (SDW) to re-suspend bacteria, elicited the slightest response in mock-inoculated plants. Under this method, Agrobacterium strains carrying the GUS reporter gene were used to test bacterial infectivity. Blue sectors were found in plants infected for 24 and 48 h. PR1 and NPR1 expression were significantly altered at various time intervals after inoculation. So, for experiments involving Arabidopsis-Agrobacterium interaction with minimal background influences, such as gene expression and epigenetic analyses, the flood inoculation method using SDW as the resuspension liquid is proposed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01381-x.

Epimutations and mutations, nurturing phenotypic diversity.

Epimutations and mutations are two dissimilar mechanisms that have contributed to the phenotypic diversities in organisms. Though dissimilar, many previous studies have revealed that the consequences of epimutations and mutations are not mutually exclusive. DNA rich in epigenetic modifications can be prone to mutations and vice versa. In order to get a better insight into the molecular evolution in organisms, it is important to consider the information of both genetic and epigenetic changes in their genomes. Understanding the similarities and differences between the consequences of epimutations and mutations is required for a better interpretation of phenotypic diversities in organisms. Factors contributing to epigenetic changes such as paramutations and mutation hotspots and, the correlation of the interdependence of mutations and epigenetic changes in DNA are important aspects that need to be considered for molecular evolutionary studies. Thus, this review explains what epimutations are, their causes, how they are similar/different from mutations, and the influence of epigenetic changes and mutations on each other, further emphasizing how molecular evolution involving both mutations and epimutations can lead to speciation. Considering this approach will aid in reorganizing taxonomic classifications, importantly, solving disparities in species identification.

Can genetic engineering-based methods for gene function identification be eclipsed by genome editing in plants? A comparison of methodologies.

Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.

Screening internal controls for expression analyses involving numerous treatments by combining statistical methods with reference gene selection tools.

Real-time PCR is always the method of choice for expression analyses involving comparison of a large number of treatments. It is also the favored method for final confirmation of transcript levels followed by high throughput methods such as RNA sequencing and microarray. Our analysis comprised 16 different permutation and combinations of treatments involving four different Agrobacterium strains and three time intervals in the model plant Arabidopsis thaliana. The routinely used reference genes for biotic stress analyses in plants showed variations in expression across some of our treatments. In this report, we describe how we narrowed down to the best reference gene out of 17 candidate genes. Though we initiated our reference gene selection process using common tools such as geNorm, Normfinder and BestKeeper, we faced situations where these software-selected candidate genes did not completely satisfy all the criteria of a stable reference gene. With our novel approach of combining simple statistical methods such as t test, ANOVA and post hoc analyses, along with the routine software-based analyses, we could perform precise evaluation and we identified two genes, UBQ10 and PPR as the best reference genes for normalizing mRNA levels in the context of 16 different conditions of Agrobacterium infection. Our study emphasizes the usefulness of applying statistical analyses along with the reference gene selection software for reference gene identification in experiments involving the comparison of a large number of treatments.

Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains.

BACKGROUND: Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional ß-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies. RESULTS: For plants co-cultivated for 48 h with Agrobacterium, if the strain contained vir genes, then the rates of transversions, SHRs and FSMs (measured 2 weeks later) were lower than those of uninfected controls. In contrast, co-cultivation for 48 h with any of the Agrobacterium strains raised the transposition rates above control levels. The multiple time-point study showed that in seedlings co-cultivated with wild type Ach5, the reduced rates of transversions and SHRs after 48 h co-cultivation represent an apparent suppression of an earlier short-lived increase in mutation rates (peaking for plants co-cultivated for 3 h). An increase after 3 h co-cultivation was also seen for rates of transversions (but not SHR) in seedlings exposed to the strain lacking vir genes, oncogenes and T-DNA. However, the mutation rates in plants co-cultivated for longer times with this strain subsequently dropped below levels seen in uninfected controls, consistent with the results of the single time-point study. CONCLUSIONS: The rates of various classes of mutations that result from Agrobacterium infection depend upon the duration of infection and the type of pathogen derived factors (such as Vir proteins, oncoproteins or T-DNA) possessed by the strain. Strains with vir genes, including the type used for plant transformation, suppressed selected classes of somatic mutations. Our study also provides evidence of a pathogen that can at least partly counter the induction of mutations in an infected plant.

A novel T-DNA integration in rice involving two interchromosomal translocations.

A male sterile transgenic rice plant TC-19 harboured a novel T-DNA integration in chromosome 8 with two interchromosomal translocations of 6.55 kb chromosome 3 and 29.8 kb chromosome 9 segments. We report a complex Agrobacterium T-DNA integration in rice (Oryza sativa) associated with two interchromosomal translocations. The T-DNA-tagged rice mutant TC-19, which harboured a single copy of the T-DNA, displayed male sterile phenotype in the homozygous condition. Analysis of the junctions between the T-DNA ends and the rice genome by genome walking showed that the right border is flanked by a chromosome 3 sequence and the left border is flanked by a chromosome 9 sequence. Upon further walking on chromosome 3, a chromosome 3/chromosome 8 fusion was detected. Genome walking from the opposite end of the chromosome 8 break point revealed a chromosome 8/chromosome 9 fusion. Our findings revealed that the T-DNA, together with a 6.55-kb region of chromosome 3 and a 29.8-kb region of chromosome 9, was translocated to chromosome 8. Southern blot analysis of the homozygous TC-19 mutant revealed that the native sequences of chromosome 3 and 9 were restored but the disruption of chromosome 8 in the first intron of the gene Os08g0152500 was not restored. The integration of the complex T-DNA in chromosome 8 caused male sterility.

Epigenetic regulation influenced by soil microbiota and nutrients: Paving road to epigenome editing in plants.

Soil is a complex ecosystem that houses microbes and nutrients that are necessary for plant development. Edaphic properties of the soil and environmental conditions influence microbial growth and nutrient accessibility. Various environmental stimuli largely affect the soil microbes and ionic balance, in turn influencing plants. Soil microflora helps decompose organic matter and is involved in mineral uptake. The combination of soil microbes and mineral nutrients notably affects plant growth. Recent advancements have enabled a deeper understanding of plant genetic/molecular regulators. Deficiencies/sufficiencies of soil minerals and microbes also alter plant gene regulation. Gene regulation mediated by epigenetic mechanisms comprises conformational alterations in chromatin structure, DNA/histone modifications, or involvement of small RNAs. Epigenetic regulation is unique due to its potential to inherit without involving alteration of the DNA sequence. Thus, the compilation study of heritable epigenetic changes driven by nutrient imbalances and soil microbes would facilitate understanding this molecular phenomenon in plants. This information can aid in epigenome editing, which has recently emerged as a promising technology for plant non-transgenic/non-mutagenic modification. Potential epigenetic marks induced by biotic and abiotic stresses in plants could be explored as target sites for epigenome editing. This review discusses novel ways of epigenome editing to create epigenome edited plants with desirable and heritable phenotypes. As plants are sessile and in constant exposure to the soil microbiome and nutrients, epigenetic changes induced by these factors could provide more effective, stable and a sustainable molecular solution for crop improvement.

Recalcitrance to transformation, a hindrance for genome editing of legumes.

Plant genome editing, a recently discovered method for targeted mutagenesis, has emerged as a promising tool for crop improvement and gene function research. Many genome-edited plants, such as rice, wheat, and tomato, have emerged over the last decade. As the preliminary steps in the procedure for genome editing involve genetic transformation, amenability to genome editing depends on the efficiency of genetic engineering. Hence, there are numerous reports on the aforementioned crops because they are transformed with relative ease. Legume crops are rich in protein and, thus, are a favored source of plant proteins for the human diet in most countries. However, legume cultivation often succumbs to various biotic/abiotic threats, thereby leading to high yield loss. Furthermore, certain legumes like peanuts possess allergens, and these need to be eliminated as these deprive many people from gaining the benefits of such crops. Further genetic variations are limited in certain legumes. Genome editing has the potential to offer solutions to not only combat biotic/abiotic stress but also generate desirable knock-outs and genetic variants. However, excluding soybean, alfalfa, and Lotus japonicus, reports obtained on genome editing of other legume crops are less. This is because, excluding the aforementioned three legume crops, the transformation efficiency of most legumes is found to be very low. Obtaining a higher number of genome-edited events is desirable as it offers the option to genotypically/phenotypically select the best candidate, without the baggage of off-target mutations. Eliminating the barriers to genetic engineering would directly help in increasing genome-editing rates. Thus, this review aims to compare various legumes for their transformation, editing, and regeneration efficiencies and discusses various solutions available for increasing transformation and genome-editing rates in legumes.

Essential role of the BRCA2B gene in somatic homologous recombination in Arabidopsis thaliana.

Constant exposure to various environmental and endogenous stresses can cause structural DNA damage, resulting in genome instability. Higher eukaryotic cells deploy conserved DNA repair systems, which include various DNA repair pathways, to maintain genome stability. Homologous recombination (HR), one of these repair pathways, involves multiple proteins. BRCA2, one of the proteins in the HR pathway, is of substantial research interest in humans because it is an oncogene. However, the study of this gene is limited due to the lack of availability of homozygous BRCA2-knockout mutants in mammals, which results in embryonic lethality. Arabidopsis thaliana has two copies of the BRCA2 homologs: BRCA2A and BRCA2B . Therefore, the single mutants remain nonlethal and fertile in Arabidopsis. The BRCA2A homolog, which plays a significant role in the HR pathway of germline cells and during the defense response, is well-studied in Arabidopsis. Our study focuses on the functional characterization of the BRCA2B homolog in the somatic cells of Arabidopsis, using the homozygous ΔBRCA2B mutant line. The phenotypic differences of ΔBRCA2B mutants were characterized and compared with wild Arabidopsis plants. The role of BRCA2B in spontaneous somatic HR (SHR) was studied using the ΔBRCA2B-gus detector line. ΔBRCA2B plants have a 6.3-fold lower SHR frequency than the control detector plants. Expression of four other HR pathway genes, including BRE, BRCC36A, RAD50, and RAD54, was significantly reduced in ΔBRCA2B mutants. Thus, our findings convey that the BRCA2B homolog plays an important role in maintaining spontaneous SHR rates and has a direct or indirect regulatory effect on the expression of other HR-related genes.

Enhanced sheath blight resistance in transgenic rice expressing an endochitinase gene from Trichoderma virens.

The 42-kDa endochitinase (cht42) gene from the mycoparasitic fungus, Trichoderma virens, driven by CaMV 35S promoter, was introduced into rice by Agrobacterium-mediated transformation. Eight transgenic plants harboring single copies of complete T-DNA were identified by Southern blot analysis. Homozygous transgenic plants were identified for five lines in the T(1) generation by Southern blot analysis. Homozygous T(2) plants constitutively accumulated high levels of the cht42 transcript, showed 2.4- to 4.6-fold higher chitinase activity in total leaf extract and 1.6- to 1.8-fold higher chitinase activity in the extracellular fluid. Infection assays performed on the homozygous T(2) plants with Rhizoctonia solani showed up to 62% reduction in the sheath blight disease index.

Plant reference genes for development and stress response studies.

Many reference genes are used by different laboratories for gene expression analyses to indicate the relative amount of input RNA/DNA in the experiment. These reference genes are supposed to show least variation among the treatments and with the control sets in a given experiment. However, expression of reference genes varies significantly from one set of experiment to the other. Thus, selection of reference genes depends on the experimental conditions. Sometimes the average expression of two or three reference genes is taken as standard. This review consolidated the details of about 120 genes attempted for normalization during comparative expression analysis in 16 different plants. Plant species included in this review are Arabidopsis thaliana, cotton (Gossypium hirsutum), tobacco (Nicotiana benthamiana and N. tabacum), soybean (Glycine max), rice (Oryza sativa), blueberry (Vaccinium corymbosum), tomato (Solanum lycopersicum), wheat (Triticum aestivum), potato (Solanum tuberosum), sugar cane (Saccharum sp.), carrot (Daucus carota), coffee (Coffea arabica), cucumber (Cucumis sativus), kiwi (Actinidia deliciosa) and grape (Vitis vinifera). The list includes model and cultivated crop plants from both monocot and dicot classes. We have categorized plant-wise the reference genes that have been used for expression analyses in any or all of the four different conditions such as biotic stress, abiotic stress, developmental stages and various organs and tissues, reported till date. This review serves as a guide during the reference gene hunt for gene expression analysis studies.