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.

High- or Low-Yielding F2 Progeny of Wheat Is Result of Specific TaCKX Gene Coexpression Patterns in Association with Grain Yield in Paternal Parent.

Members of the TaCKX gene family (GFM) encode oxidase/dehydrogenase cytokinin degrading enzymes (CKX), which play an important role in the homeostasis of phytohormones, affecting wheat development and productivity. Therefore, the objective of this investigation was to test how the expression patterns of the yield-related TaCKX genes and TaNAC2-5A (NAC2) measured in 7 days after pollination (DAP) spikes and the seedling roots of parents are inherited to apply this knowledge in the breeding process. The expression patterns of these genes were compared between parents and their F2 progeny in crosses of one mother with different paterns of awnless cultivars and reciprocal crosses of awned and awnless lines. We showed that most of the genes tested in the 7 DAP spikes and seedling roots of the F2 progeny showed paternal expression patterns in crosses of awnless cultivars as well as reciprocal crosses of awned and awnless lines. Consequently, the values of grain yield in the F2 progeny were similar to the pater; however, the values of seedling root mass were similar to the mother or both parents. The correlation analysis of TaCKX GFMs and NAC2 in spikes and spikes per seedling roots reveals that the genes correlate with each other specifically with the pater and the F2 progeny or the mother and the F2 progeny, which shape phenotypic traits. The numbers of spikes and semi-empty spikes are mainly correlated with the specific coexpression of the TaCKX and NAC2 genes expressed in spikes or spikes per roots of the pater and F2 progeny. Variable regression analysis of grain yield and root mass with TaCKX GFMs and NAC2 expressed in the tested tissues of five crosses revealed a significant dependency of these parameters on the mother and F2 and/or the pater and F2 progeny. We showed that the inheritance of yield-related traits depends on the specific cooperative expression of some TaCKX GFMs, in some crosses coupled with NAC2, and is strongly dependent on the genotypes used for the crosses. Indications for parental selection in the breeding of high-yielding lines are discussed.

HvGSK1.1 Controls Salt Tolerance and Yield through the Brassinosteroid Signaling Pathway in Barley.

Brassinosteroids (BRs) are a class of plant steroid hormones that are essential for plant growth and development. BRs control important agronomic traits and responses to abiotic stresses. Through the signaling pathway, BRs control the expression of thousands of genes, resulting in a variety of biological responses. The key effectors of the BR pathway are two transcription factors (TFs): BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMSSUPPRESSOR 1 (BES1). Both TFs are phosphorylated and inactivated by the Glycogen synthase kinase 3 BRASSINOSTEROID INSENSITIVE2 (BIN2), which acts as a negative regulator of the BR pathway. In our study, we describe the functional characteristics of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. We generated mutant lines of HvGSK1.1 using CRISPR/Cas9 genome editing technology. Next Generation Sequencing (NGS) of the edited region of the HvGSK1.1 showed a wide variety of mutations. Most of the changes (frameshift, premature stop codon, and translation termination) resulted in the knock-out of the target gene. The molecular and phenotypic characteristics of the mutant lines showed that the knock-out mutation of HvGSK1.1 improved plant growth performance under salt stress conditions and increased the thousand kernel weight of the plants grown under normal conditions. The inactivation of HvGSK1.1 enhanced BR-dependent signaling, as indicated by the results of the leaf inclination assay in the edited lines. The plant traits under investigation are consistent with those known to be regulated by BRs. These results, together with studies of other GSK3 gene members in other plant species, suggest that targeted editing of these genes may be useful in creating plants with improved agricultural traits.

Low Molecular Weight and High Deacetylation Degree Chitosan Batch Alleviates Pathogenesis, Toxin Accumulation, and Fusarium Gene Regulation in Barley Leaf Pathosystem.

Fusarium graminearum is a cosmopolitan fungal pathogen that destroys cereal production, in terms of loss of yield and grain contamination with mycotoxins, worldwide. Chitosan is a natural biopolymer abundant in the environment with proven antifungal properties that also acts as a plant immunity elicitor. Despite a number of articles, there is a lack of systematic comparison of antifungal activity of diverse batches of chitosan. The current study aimed to test the inhibitory effects of a collection of diverse chitosan samples on the growth and production of F. graminearum toxins, validated by changes in the Fusarium transcriptome. Experiments included testing antifungal activity of different chitosan samples, the application of the best performing one in vitro to investigate the impact on F. graminearum growth, followed by analyzing its effect on Fusarium toxins accumulation, and Fusarium transcriptomics in the barley leaf pathosystem. Confirmatory antifungal assays revealed that CS_10, a specific batch of chitosan, retarded Fusarium growth with an application concentration of 200 ppm, significantly reducing toxin synthesis and disease symptoms in Fusarium-inoculated barley leaves. RNA-Seq analysis of F. graminearum in barley leaf pathosystem exposed to CS_10 showed a list of differentially expressed genes involved in redox balance, cell respiration, nutrient transport, cell wall degradation enzymes, ergosterol biosynthesis, and trichothecenes production. The genes functioning in these essential pathways are discussed and assigned as critical checkpoints to control Fusarium infections. The results suggest some important molecular targets in F. graminearum that may be suitable in gene-specific targeting or transgene-free methods, such as spray-induced gene silencing during host-pathogen interactions.

Transgenerational Paternal Inheritance of TaCKX GFMs Expression Patterns Indicate a Way to Select Wheat Lines with Better Parameters for Yield-Related Traits.

Members of the TaCKX gene family (GFMs) encode the cytokinin oxygenase/dehydrogenase enzyme (CKX), which irreversibly degrades cytokinins in the organs of wheat plants; therefore, these genes perform a key role in the regulation of yield-related traits. The purpose of the investigation was to determine how expression patterns of these genes, together with the transcription factor-encoding gene TaNAC2-5A, and yield-related traits are inherited to apply this knowledge to speed up breeding processes. The traits were tested in 7 days after pollination (DAP) spikes and seedling roots of maternal and paternal parents and their F2 progeny. The expression levels of most of them and the yield were inherited in F2 from the paternal parent. Some pairs or groups of genes cooperated, and some showed opposite functions. Models of up- or down-regulation of TaCKX GFMs and TaNAC2-5A in low-yielding maternal plants crossed with higher-yielding paternal plants and their high-yielding F2 progeny reproduced gene expression and yield of the paternal parent. The correlation coefficients between TaCKX GFMs, TaNAC2-5A, and yield-related traits in high-yielding F2 progeny indicated which of these genes were specifically correlated with individual yield-related traits. The most common was expressed in 7 DAP spikes TaCKX2.1, which positively correlated with grain number, grain yield, spike number, and spike length, and seedling root mass. The expression levels of TaCKX1 or TaNAC2-5A in the seedling roots were negatively correlated with these traits. In contrast, the thousand grain weight (TGW) was negatively regulated by TaCKX2.2.2, TaCKX2.1, and TaCKX10 in 7 DAP spikes but positively correlated with TaCKX10 and TaNAC2-5A in seedling roots. Transmission of TaCKX GFMs and TaNAC2-5A expression patterns and yield-related traits from parents to the F2 generation indicate their paternal imprinting. These newly shown data of nonmendelian epigenetic inheritance shed new light on crossing strategies to obtain a high-yielding F2 generation.

Polyamine Oxidation Is Indispensable for Wheat (Triticum aestivum L.) Oxidative Response and Necrotic Reactions during Leaf Rust (Puccinia triticina Eriks.) Infection.

Hydrogen peroxide is a signal and effector molecule in the plant response to pathogen infection. Wheat resistance to Puccinia triticina Eriks. is associated with necrosis triggered by oxidative burst. We investigated which enzyme system dominated in host oxidative reaction to P. triticina infection. The susceptible Thatcher cultivar and isogenic lines with defined resistance genes were inoculated with P. triticina spores. Using diamine oxidase (DAO) and polyamine oxidase (PAO) inhibitors, accumulation of H2O2 was analyzed in the infection sites. Both enzymes participated in the oxidative burst during compatible and incompatible interactions. Accumulation of H2O2 in guard cells, i.e., the first phase of the response, depended on DAO and the role of PAO was negligible. During the second phase, the patterns of H2O2 accumulation in the infection sites were more complex. Accumulation of H2O2 during compatible interaction (Thatcher and TcLr34 line) moderately depended on DAO and the reaction of TcLr34 was stronger than that of Thatcher. Accumulation of H2O2 during incompatible interaction of moderately resistant plants (TcLr24, TcLr25 and TcLr29) was DAO-dependent in TcLr29, while the changes in the remaining lines were not statistically significant. A strong oxidative burst in resistant plants (TcLr9, TcLr19, TcLr26) was associated with both enzymes' activities in TcLr9 and only with DAO in TcLr19 and TcLr26. The results are discussed in relation to other host oxidative systems, necrosis, and resistance level.

Different sets of TaCKX genes affect yield-related traits in wheat plants grown in a controlled environment and in field conditions.

BACKGROUND: TaCKX wheat gene family members (GFMs) encode the enzyme cytokinin oxidase/dehydrogenase (CKX), which irreversibly degrades cytokinins. The genes are important regulators of cytokinin content and take part in growth and development, with a major impact on yield-related traits. The goal of this research was to test whether these genes might be differentially expressed in the field compared to laboratory conditions and consequently differently affect plant development and yield. RESULTS: We compared expression and crosstalk of the TaCKX GFMs and TaNAC2-5A gene in modern varieties grown in a growth chamber (GC) and in the field and looked for differences in their impact on yield-related traits. The TaNAC2-5A gene was included in the research since it was expected to play an important role in co-regulation of these genes. The range of relative expression levels of TaCKX GFMs and TaNAC2-5A gene among tested cultivars was from 5 for TaCKX8 to more than 100 for TaCKX9 in the GC and from 6 for TaCKX8 to 275 for TaCKX10 in the field. The range was similar for four of them in the GC, but was much higher for seven others and TaNAC2-5A in the field. The TaCKX GFMs and TaNAC2-5A form co-expression groups, which differ depending on growth conditions. Consequently, the genes also differently regulate yield-related traits in the GC and in the field. TaNAC2-5A took part in negative regulation of tiller number and CKX activity in seedling roots only in controlled GC conditions. Grain number and grain yield were negatively regulated by TaCKX10 in the GC but positively by TaCKX8 and others in the field. Some of the genes, which were expressed in seedling roots, negatively influenced tiller number and positively regulated seedling root weight, CKX activity in the spikes, thousand grain weight (TGW) as well as formation of semi-empty spikes. CONCLUSIONS: We have documented that: 1) natural variation in expression levels of tested genes in both environments is very high, indicating the possibility of selection of beneficial genotypes for breeding purposes, 2) to create a model of an ideotype for breeding, we need to take into consideration the natural environment.

Silencing of HvGSK1.1-A GSK3/SHAGGY-Like Kinase-Enhances Barley (Hordeum vulgare L.) Growth in Normal and in Salt Stress Conditions.

Glycogen synthase kinase 3 (GSK3) is a highly conserved kinase present in all eukaryotes and functions as a key regulator of a wide range of physiological and developmental processes. The kinase, known in land plants as GSK3/SHAGGY-like kinase (GSK), is a key player in the brassinosteroid (BR) signaling pathway. The GSK genes, through the BRs, affect diverse developmental processes and modulate responses to environmental factors. In this work, we describe functional analysis of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. The RNAi-mediated silencing of the target HvGSK1.1 gene was associated with modified expression of its paralogs HvGSK1.2, HvGSK2.1, HvGSK3.1, and HvGSK4.1 in plants grown in normal and in salt stress conditions. Low nucleotide similarity between the silencing fragment and barley GSK genes and the presence of BR-dependent transcription factors' binding sites in promoter regions of barley and rice GSK genes imply an innate mechanism responsible for co-regulation of the genes. The results of the leaf inclination assay indicated that silencing of HvGSK1.1 and the changes of GSK paralogs enhanced the BR-dependent signaling in the plants. The strongest phenotype of transgenic lines with downregulated HvGSK1.1 and GSK paralogs had greater biomass of the seedlings grown in normal conditions and salt stress as well as elevated kernel weight of plants grown in normal conditions. Both traits showed a strong negative correlation with the transcript level of the target gene and the paralogs. The characteristics of barley lines with silenced expression of HvGSK1.1 are compatible with the expected phenotypes of plants with enhanced BR signaling. The results show that manipulation of the GSK-encoding genes provides data to explore their biological functions and confirm it as a feasible strategy to generate plants with improved agricultural traits.

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.

TaWAK6 encoding wall-associated kinase is involved in wheat resistance to leaf rust similar to adult plant resistance.

In wheat, adult plant resistance (APR) to leaf rust (Puccinia triticina), is effective in restricting pathogen growth and provides durable resistance against a wide range of virulent forms of P. triticina. Despite the importance, there is limited knowledge on the molecular basis of this type of resistance. We isolated and characterized the wall-associated kinase encoding gene in wheat, and assigned it as TaWAK6. Localization of TaWAK6 homeologs in A and B wheat subgenomes was consistent with the presence of the gene's orthologs in T. urartu (AA) and T. dicoccoides (AABB) and with the absence of its orthologs in Aegilops tauschii (DD). Overexpression of TaWAK6 did not change the wheat phenotype, nor did it affect seedling resistance. However, the adult plants overexpressing TaWAK6 showed that important parameters of APR were significantly elevated. Infection types scored on the first (flag), second and third leaves indicated elevated resistance, which significantly correlated with expression of TaWAK6. Analysis of plant-pathogen interactions showed a lower number of uredinia and higher rates of necrosis at the infection sites and this was associated with smaller size of uredinia and a longer latent period. The results indicated a role of TaWAK6 in quantitative partial resistance similar to APR in wheat. It is proposed that TaWAK6, which is a non-arginine-aspartate (non-RD) kinase, represents a novel class of quantitative immune receptors in monocots.

Specificity of expression of TaCKX family genes in developing plants of wheat and their co-operation within and among organs.

Multigene families of CKX genes encode cytokinin oxidase/dehydrogenase proteins (CKX), which regulate cytokinin content in organs of developing plants. It has already been documented that some of them play important roles in plant productivity. The presented research is the first step of comprehensive characterization of the bread wheat TaCKX gene family with the goal to select genes determining yield-related traits. The specificity of expression patterns of fifteen formerly annotated members of the TaCKX family was tested in different organs during wheat development. Based on this, the genes were assigned to four groups: TaCKX10, TaCKX5 and TaCKX4, highly specific to leaves; TaCKX3, TaCKX6 and TaCKX11, expressed in various levels through all the organs tested; TaCKX1, TaCKX2.3, TaCKX2.2, TaCKX2.1, TaCKX2.4 and TaCKX2.5 specific to developing spikes and inflorescences; TaCKX9, TaCKX8 and TaCKX7, highly specific to roots. Amplification products of tested genes were mapped to the chromosomes of the A, B or D genome using T. aestivum Ensembl Plants. Based on analysis of TaCKX transcripts as well as encoded amino acids in T. aestivum and Hordeum vulgare the number of CKX genes in wheat was limited to 11 and new numbering of selected TaCKX genes was proposed. Moreover, we found that there were developmental differences in expression of TaCKX in the first and the second spike and expression of some of the genes was daily time dependent. A very high and significant correlation was found between expression levels of TaCKX7 and TaCKX9, genes specific to seedling roots, TaCKX1, TaCKX2.1 and TaCKX2.2, specific to developing spikes, and the group of TaCKX3, 4, 5, 6, 10 and 11, highly expressed in leaves and other organs. The genes also co-operated among organs and were included in two groups representing younger or maturating stages of developing plants. The first group was represented by seedling roots, leaves from 4-week old plants, inflorescences and 0 DAP spikes; the second by developing spikes, 0 DAP, 7 DAP and 14 DAP. The key genes which might determine yield-related traits are indicated and their possible roles in breeding strategies are discussed.

Pathogen-regulated genes in wheat isogenic lines differing in resistance to brown rust Puccinia triticina.

BACKGROUND: Inoculation of wheat plants with Puccinia triticina (Pt) spores activates a wide range of host responses. Compatible Pt interaction with susceptible Thatcher plants supports all stages of the pathogen life cycle. Incompatible interaction with TcLr9 activates defense responses including oxidative burst and micronecrotic reactions associated with the pathogen's infection structures and leads to complete termination of pathogen development. These two contrasting host-pathogen interactions were a foundation for transcriptome analysis of incompatible wheat-Pt interaction. METHODS: A suppression subtractive hybridization (SSH) library was constructed using cDNA from pathogen-inoculated susceptible Thatcher and resistant TcLr9 isogenic lines. cDNA represented steps of wheat-brown rust interactions: spore germination, haustorium mother cell (HMC) formation and micronecrotic reactions. All ESTs were clustered and validated by similarity search to wheat genome using BLASTn and sim4db tools. qRT-PCR was used to determine transcript levels of selected ESTs after inoculation in both lines. RESULTS AND DISCUSSION: Out of 793 isolated cDNA clones, 183 were classified into 152 contigs. 89 cDNA clones and encoded proteins were functionally annotated and assigned to 5 Gene Ontology categories: catalytic activity 48 clones (54 %), binding 32 clones (36 %), transporter activity 6 clones (7 %), structural molecule activity 2 clones (2 %) and molecular transducer activity 1 clone (1 %). Detailed expression profiles of 8 selected clones were analyzed using the same plant-pathogen system. The strongest induction after pathogen infection and the biggest differences between resistant and susceptible interactions were detected for clones encoding wall-associated kinase (GenBank accession number JG969003), receptor with leucine-rich repeat domain (JG968955), putative serine/threonine protein kinase (JG968944), calcium-mediated signaling protein (JG968925) and 14-3-3 protein (JG968969). CONCLUSIONS: The SSH library represents transcripts regulated by pathogen infection during compatible and incompatible interactions of wheat with P. triticina. Annotation of selected clones confirms their putative roles in successive steps of plant-pathogen interactions. The transcripts can be categorized as defense-related due to their involvement in either basal defense or resistance through an R-gene mediated reaction. The possible involvement of selected clones in pathogen recognition and pathogen-induced signaling as well as resistance mechanisms such as cell wall enforcement, oxidative burst and micronecrotic reactions is discussed.

A new BSMV-based vector with modified ß molecule allows simultaneous and stable silencing of two genes.

Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:ß molecule was engineered with a unique BamHI site between the open reading frame of ßc (ORF ßc) and poly(A). The mixture of RNA particles α, ßBamHI and γMCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to ßBamHI and γMCS. Delivery of the target gene fragment in γMCS induced stronger silencing, while delivery in ßBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of ß enables simultaneous silencing of two genes. Quantifying the ß and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than ß, while the stability of the insert was higher in ß compared with γ. The possible influence of the relative quantity of ß and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.