Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

Identification of quantitative trait loci associated with leaf rust resistance in rye by precision mapping.

BACKGROUND: Leaf rust (LR) is among the most destructive fungal diseases of rye (Secale cereale L.). Despite intensive research using various analytical and methodological approaches, such as quantitative trait locus (QTL) mapping, candidate gene expression analysis, and transcriptome sequencing, the genetic basis of the rye immune response to LR remains unclear. RESULTS: A genome-wide association study was employed to detect QTLs controlling the immune response to LR of rye. A mapping population, G38A, was constructed by crossing two inbred lines: 723 (susceptible to LR) and JKI-NIL-Pr3 (a donor of the LR resistance gene Pr3). For genotyping, SNP-DArT and silico-DArT markers were used. Resistance phenotyping was conducted by visual assessment of the infection severity in detached leaf segments inoculated with two isolates of Puccinia recondita f. sp. secalis, namely, 60/17/2.1 (isolate S) in the main experiment and 86/n/2.1_5x (isolate N) in the validation experiment, at 10 and 17 days post-infection (dpi), respectively. In total, 42,773 SNP-DArT and 105,866 silico-DArT markers were included in the main analysis including isolate S, of which 129 and 140 SNP-DArTs and 767 and 776 silico-DArTs were significantly associated (p ≤ 0.001; - log10(p) ≥ 3.0) with the immune response to LR at 10 and 17 dpi, respectively. Most significant markers were mapped to chromosome 1R. The number of common markers from both systems and at both time points occupying common chromosomal positions was 37, of which 21 were positioned in genes, comprising 18 markers located in exons and three in introns. This gene pool included genes encoding proteins with a known function in response to LR (e.g., a NBS-LRR disease resistance protein-like protein and carboxyl-terminal peptidase). CONCLUSION: This study has expanded and supplemented existing knowledge of the genetic basis of rye resistance to LR by (1) detecting two QTLs associated with the LR immune response of rye, of which one located on the long arm of chromosome 1R is newly detected, (2) assigning hundreds of markers significantly associated with the immune response to LR to genes in the 'Lo7' genome, and (3) predicting the potential translational effects of polymorphisms of SNP-DArT markers located within protein-coding genes.

Genotype-Specific Expression of Selected Candidate Genes Conferring Resistance to Leaf Rust of Rye (Secale cereale L.).

Leaf rust (LR) caused by Puccinia recondita f. sp. secalis (Prs) is a highly destructive disease in rye. However, the genetic mechanisms underlying the rye immune response to this disease remain relatively uncharacterised. In this study, we analysed the expression of four genes in 12 rye inbred lines inoculated with Prs at 20 and 36 h post-treatment (hpt): DXS (1-deoxy-D-xylulose 5-phosphate synthase), Glu (ß-1,3-glucanase), GT (UDP-glycosyltransferase) and PR-1 (pathogenesis-related protein 1). The RT-qPCR analysis revealed the upregulated expression of the four genes in response to Prs in all inbred lines and at both time-points. The gene expression data were supported by microscopic and macroscopic examinations, which revealed that eight lines were susceptible to LR and four lines were highly resistant to LR. A relationship between the infection profiles and the expression of the analysed genes was observed: in the resistant lines, the expression level fold changes were usually higher at 20 hpt than at 36 hpt, while the opposite trend was observed in the susceptible lines. The study results indicate that DXS, Glu, GT and PR-1 may encode proteins crucial for the rye defence response to the LR pathogen.

Identification and characteristics of wheat Lr orthologs in three rye inbred lines.

The genetic background of the immune response of rye to leaf rust (LR), although extensively studied, is still not well understood. The recent publication of the genome of rye line Lo7 and the development of efficient transcriptomic methods has aided the search for genes that confer resistance to this disease. In this study, we investigated the potential role of rye orthologs of wheat Lr genes (Lr1, Lr10, Lr21, Lr22a, and RGA2/T10rga2-1A) in the LR seedling-stage resistance of inbred rye lines D33, D39, and L318. Bioinformatics analysis uncovered numerous Lr orthologs in the Lo7 genome, namely, 14 ScLr1, 15 ScRga2, and 2 ScLr21 paralogs, and 1 each of ScLr10 and ScLr22a genes. The paralogs of ScLr1, ScRga2, and ScLr21 were structurally different from one another and their wheat counterparts. According to an RNA sequencing analysis, only four wheat Lr gene orthologs identified in the Lo7 genome (ScLr1_3, ScLr1_4, ScLr1_8, and ScRga2_6) were differentially expressed; all four were downregulated after infection with compatible or incompatible isolates of Puccinia recondita f. sp. secalis (Prs). Using a more precise tool, RT-qPCR, we found that two genes were upregulated at 20 h post-infection, namely, ScLr1_4 and ScLr1_8 in lines D33 and D39, respectively, both of which have been found to be resistant to LR under field conditions and after treatment with a semi-compatible Prs strain. We were unable to discern any universal pattern of gene expression after Prs infection; on the contrary, all detected relationships were plant genotype-, Prs isolate-, or time-specific. Nevertheless, at least some Lr orthologs in rye (namely, ScLr1_3 ScLr1_4, ScLr1_8, and ScRga2_6), even though mainly downregulated, may play an important role in the response of rye to LR.

Identification of candidate genes responsible for chasmogamy in wheat.

BACKGROUND: The flowering biology of wheat plants favours self-pollination which causes obstacles in wheat hybrid breeding. Wheat flowers can be divided into two groups, the first one is characterized by flowering and pollination within closed flowers (cleistogamy), while the second one possesses the ability to open flowers during processes mentioned above (chasmogamy). The swelling of lodicules is involved in the flowering of cereals and among others their morphology, calcium and potassium content differentiate between cleistogamic and non-cleistogamous flowers. A better understanding of the chasmogamy mechanism can lead to the development of tools for selection of plants with the desired outcrossing rate. To learn more, the sequencing of transcriptomes (RNA-Seq) and Representational Difference Analysis products (RDA-Seq) were performed to investigate the global transcriptomes of wheat lodicules in two highly chasmogamous (HCH, Piko and Poezja) and two low chasmogamous (LCH, Euforia and KWS Dacanto) varieties at two developmental stages-pre-flowering and early flowering. RESULTS: The differentially expressed genes were enriched in five, main pathways: "metabolism", "organismal systems", "genetic information processing", "cellular processes" and "environmental information processing", respectively. Important genes with opposite patterns of regulation between the HCH and LCH lines have been associated with the lodicule development i.e. expression levels of MADS16 and MADS58 genes may be responsible for quantitative differences in chasmogamy level in wheat. CONCLUSIONS: We conclude that the results provide a new insight into lodicules involvement in the wheat flowering process. This study generated important genomic information to support the exploitation of the chasmogamy in wheat hybrid breeding programs.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100-200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Identification of Rf Genes in Hexaploid Wheat (Triticumaestivum L.) by RNA-Seq and Paralog Analyses.

Among the natural mechanisms used for wheat hybrid breeding, the most desirable is the system combining the cytoplasmic male sterility (cms) of the female parent with the fertility-restoring genes (Rf) of the male parent. The objective of this study was to identify Rf candidate genes in the wheat genome on the basis of transcriptome sequencing (RNA-seq) and paralog analysis data. Total RNA was isolated from the anthers of two fertility-restorer (Primépi and Patras) and two non-restorer (Astoria and Grana) varieties at the tetrad and late uninucleate microspore stages. Of 36,912 differentially expressed genes (DEGs), 21 encoding domains in known fertility-restoring proteins were selected. To enrich the pool of Rf candidates, 52 paralogs (PAGs) of the 21 selected DEGs were included in the analyses. The expression profiles of most of the DEGs and PAGs determined bioinformatically were as expected (i.e., they were overexpressed in at least one fertility-restorer variety). However, these results were only partially consistent with the quantitative real-time PCR data. The DEG and PAG promoters included cis-regulatory elements common among PPR-encoding genes. On the basis of the obtained results, we designated seven genes as Rf candidate genes, six of which were identified for the first time in this study.

The Roots of Rye (Secale cereale L.) Are Capable of Synthesizing Benzoxazinoids.

According to current opinion, the first step of benzoxazinoids (BXs) synthesis, that is, the conversion of indole-3-glycerol phosphate to indole, occurs exclusively in the photosynthesising parts of plants. However, the results of our previous work and some other studies suggest that this process may also occur in the roots. In this study, we provide evidence that the first step of BXs synthesis does indeed occur in the roots of rye seedlings. We detected ScBx1 transcripts, BX1 enzyme, and six BXs (2-hydroxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one, (2R)-2-O-ß-d-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one glucoside, 2,4-dihydroxy- 7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside, and 6-methoxy-2-benzoxazolinone) in the roots developed from seeds deprived of the coleoptile at 2 days after sowing (i.e., roots without contact with aerial parts). In roots regenerated in vitro, both ScBx1 transcripts and BX1 enzyme were detected at a low but still measurable levels. Thus, BXs are able to be synthesised in both the roots and above-ground parts of rye plants.

Changes in benzoxazinoid contents and the expression of the associated genes in rye (Secale cereale L.) due to brown rust and the inoculation procedure.

Benzoxazinoids (BXs) are secondary metabolites with diverse functions, but are primarily involved in protecting plants, mainly from the family Poaceae, against insects and fungal pathogens. Rye is a cereal crop that is highly resistant to biotic stresses. However, its susceptibility to brown rust caused by Puccinia recondita f. sp. secalis (Prs) is still a major problem affecting its commercial production. Additionally, the genetic and metabolic factors related to this disease remain poorly characterized. In this study, we investigated whether and to what extent the brown rust infection and the inoculation procedure affect the contents of specific BXs (HBOA, GDIBOA, DIBOA, GDIMBOA, DIMBOA, and MBOA) and the expression of genes related to BX (ScBx1-5, ScIgl, and Scglu). We revealed that treatments with water and a urediniospore suspension usually downregulate gene expression levels. Moreover, HBOA and DIBOA contents decreased, whereas the contents of the remaining metabolites increased. Specifically, the MBOA content increased more after the mock treatment than after the Prs treatment, whereas the increase in GDIBOA and GDIMBOA levels was usually due to the Prs infection, especially at two of the most critical time-points, 17 and 24 h post-treatment. Therefore, GDIBOA and GDIMBOA are glucosides that are important components of rye defence responses to brown rust. Furthermore, along with MBOA, they protect rye against the stress associated with the inoculation procedure used in this study.

Genes ScBx1 and ScIgl-Competitors or Cooperators?

Two genes, Bx1 and Igl, both encoding indole-3-glycerol phosphate lyase (IGL), are believed to control the conversion of indole-3-glycerol phosphate (IGP) to indole. The first of these has generally been supposed to be regulated developmentally, being expressed at early stages of plant development with the indole being used in the benzoxazinoid (BX) biosynthesis pathway. In contrast, it has been proposed that the second one is regulated by stresses and that the associated free indole is secreted as a volatile. However, our previous results contradicted this. In the present study, we show that the ScIgl gene takes over the role of ScBx1 at later developmental stages, between the 42nd and 70th days after germination. In the majority of plants with silenced ScBx1 expression, ScIgl was either expressed at a significantly higher level than ScBx1 or it was the only gene with detectable expression. Therefore, we postulate that the synthesis of indole used in BX biosynthesis in rye is controlled by both ScBx1 and ScIgl, which are both regulated developmentally and by stresses. In silico and in vivo analyses of the promoter sequences further confirmed our hypothesis that the roles and modes of regulation of the ScBx1 and ScIgl genes are similar.

The miRNAome dynamics during developmental and metabolic reprogramming of tomato root infected with potato cyst nematode.

Cyst-forming plant-parasitic nematodes are pests threatening many crops. By means of their secretions cyst nematodes induce the developmental and metabolic reprogramming of host cells that lead to the formation of a syncytium, which is the sole food source for growing nematodes. The in depth micro RNA (miRNA) dynamics in the syncytia induced by Globodera rostochiensis in tomato roots was studied. The miRNAomes were obtained from syncytia covering the early and intermediate developmental stages, and were the subject of differential expression analysis. The expression of 1235 miRNAs was monitored. The fold change (log2FC) ranged from -7.36 to 8.38, indicating that this transcriptome fraction was very variable. Moreover, we showed that the DE (differentially expressed) miRNAs do not fully overlap between the selected time points, suggesting infection stage specific regulation by miRNA. The correctness of RNA-seq expression profiling was confirmed by qRT-PCR (quantitative Real Time Polymerase Chain Reaction) for seven miRNA species. Down- and up-regulated miRNA species, including their isomiRs, were further used to identify their potential targets. Among them there are a large number of transcription factors linked to different aspects of plant development belonging to gene families, such as APETALA2 (AP2), SQUAMOSA (MADS-box), MYB, GRAS, and AUXIN RESPONSE FACTOR (ARF). The substantial portion of potential target genes belong to the NB-LRR and RLK (RECEPTOR-LIKE KINASE) families, indicating the involvement of miRNA mediated regulation in defense responses. We also collected the evidence for target cleavage in the case of 29 miRNAs using one of three alternative methods: 5' RACE (5' Rapid Amplification of cDNA Ends), a search of tasiRNA within our datasets, and the meta-analysis of tomato degradomes in the GEO (Gene Expression Omnibus) database. Eight target transcripts showed a negative correlation with their respective miRNAs at two or three time points. These results indicate a large regulatory potential for miRNAs in tuning the development and defense responses.

The suppression of tomato defence response genes upon potato cyst nematode infection indicates a key regulatory role of miRNAs.

Potato cyst nematode Globodera rostochiensis is an obligate parasite of solanaceous plants, triggering metabolic and morphological changes in roots which may result in substantial crop yield losses. Previously, we used the cDNA-AFLP to study the transcriptional dynamics in nematode infected tomato roots. Now, we present the rescreening of already published, upregulated transcript-derived fragment dataset using the most current tomato transcriptome sequences. Our reanalysis allowed to add 54 novel genes to 135, already found as upregulated in tomato roots upon G. rostochiensis infection (in total - 189). We also created completely new catalogue of downregulated sequences leading to the discovery of 76 novel genes. Functional classification of candidates showed that the 'wound, stress and defence response' category was enriched in the downregulated genes. We confirmed the transcriptional dynamics of six genes by qRT-PCR. To place our results in a broader context, we compared the tomato data with Arabidopsis thaliana, revealing similar proportions of upregulated and downregulated genes as well as similar enrichment of defence related transcripts in the downregulated group. Since transcript suppression is quite common in plant-nematode interactions, we assessed the possibility of miRNA-mediated inverse correlation on several tomato sequences belonging to NB-LRR and receptor-like kinase families. The qRT-PCR of miRNAs and putative target transcripts showed an opposite expression pattern in 9 cases. These results together with in silico analyses of potential miRNA targeting to the full repertoire of tomato R-genes show that miRNA mediated gene suppression may be a key regulatory mechanism during nematode parasitism.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

Dynamics in the tomato root transcriptome on infection with the potato cyst nematode Globodera rostochiensis.

Plant parasitic nematodes infect roots and trigger the formation of specialized feeding sites by substantial reprogramming of the developmental process of root cells. In this article, we describe the dynamic changes in the tomato root transcriptome during early interactions with the potato cyst nematode Globodera rostochiensis. Using amplified fragment length polymorphism-based mRNA fingerprinting (cDNA-AFLP), we monitored 17 600 transcript-derived fragments (TDFs) in infected and uninfected tomato roots, 1-14 days after inoculation with nematode larvae. Six hundred and twenty-four TDFs (3.5%) showed significant differential expression on nematode infection. We employed GenEST, a computer program which links gene expression profiles generated by cDNA-AFLP and databases of cDNA sequences, to identify 135 tomato sequences. These sequences were grouped into eight functional categories based on the presence of genes involved in hormone regulation, plant pathogen defence response, cell cycle and cytoskeleton regulation, cell wall modification, cellular signalling, transcriptional regulation, primary metabolism and allocation. The presence of unclassified genes was also taken into consideration. This article describes the responsiveness of numerous tomato genes hitherto uncharacterized during infection with endoparasitic cyst nematodes. The analysis of transcriptome profiles allowed the sequential order of expression to be dissected for many groups of genes and the genes to be connected with the biological processes involved in compatible interactions between the plant and nematode.