Triticina Infection Research Articles

Biochemical responses in wheat due to Puccinia triticina infection

Biochemical responses in wheat due to Puccinia triticina infection

Comparative Transcriptome Analysis Reveals the Gene Expression and Regulatory Characteristics of Broad-Spectrum Immunity to Leaf Rust in a Wheat-Agropyron cristatum 2P Addition Line.

Wheat leaf rust (caused by Puccinia triticina Erikss.) is among the major diseases of common wheat. The lack of resistance genes to leaf rust has limited the development of wheat cultivars. Wheat–Agropyron cristatum (A. cristatum) 2P addition line II-9-3 has been shown to provide broad-spectrum immunity to leaf rust. To identify the specific A. cristatum resistance genes and related regulatory pathways in II-9-3, we conducted a comparative transcriptome analysis of inoculated and uninoculated leaves of the resistant addition line II-9-3 and the susceptible cultivar Fukuhokomugi (Fukuho). The results showed that there were 66 A. cristatum differentially expressed genes (DEGs) and 1389 wheat DEGs in II-9-3 during P. triticina infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and gene set enrichment analysis (GSEA) revealed that the DEGs of II-9-3 were associated with plant–pathogen interaction, MAPK signaling pathway–plant, plant hormone signal transduction, glutathione metabolism, and phenylpropanoid biosynthesis. Furthermore, many defense-related A. cristatum genes, such as two NLR genes, seven receptor kinase-encoding genes, and four transcription factor-encoding genes, were identified. Our results indicated that the key step of resistance to leaf rust involves, firstly, the gene expression of chromosome 2P upstream of the immune pathway and, secondly, the effect of chromosome 2P on the co-expression of wheat genes in II-9-3. The disease resistance regulatory pathways and related genes in the addition line II-9-3 thus could play a critical role in the effective utilization of innovative resources for leaf rust resistance in wheat breeding.

Wheat Lysin-Motif-Containing Proteins Characterization and Gene Expression Patterns under Abiotic and Biotic Stress

Lysin motif (LysM)-containing proteins (LYPs) are important pattern recognition receptors in plants. However, the evolutionary history and characteristics of LYP genes remain largely unclear in wheat. In this study, 62 LYPs were identified at genome wide in wheat. Based on phylogenetic and domain analysis, wheat LYPs were classified into 6 subgroups (group LysMe, LysMn, LYP, LYK, LysMFbox). Syntenic analysis showed the evolution of LYP genes in wheat. RNA-seq data showed that 22 genes were not expressed at any tissue or stress stimulation period. Some LYP and LYK genes were tissue- or stage- specific. The majority of TaLYK5s, TaLYK6s, TaLYP2s and TaLysMns genes were induced under chitin, flg22 and fungal treatment. qRT-PCR analysis showed that 4 genes were upregulated during Puccinia triticina infection with a peak at 18 h post inoculation. Our findings suggested that wheat LYPs may have specific roles in response to fungal infection and provided insights into the function and characteristics of wheat LYP genes.

Physiological responses and genome-wide characterization of TaNRAMP1 gene in Mn-deficient wheat

Manganese (Mn) is an essential micronutrient for plants. This study elucidates the physiological consequences and characterization of TaNRAMP1 transporter in Mn-starved wheat. The cellular integrity, redox status, chlorophyll score, and Fv/Fm were severely affected, accompanied by decreased Mn concentration in root and shoot in Mn-deficient wheat. However, Fe concentration and root phytosiderophore release were not affected, contradicting the interactions of Fe status with Mn under Mn shortage. The genome-wide identification of TaNRAMP1 (natural resistance-associated macrophage protein 1), known as high-affinity Mn transporter, showed several polymorphisms within genome A, B, and D. The expression of TaNRAMP1 significantly decreased in roots of genome A and B but was constitutively expressed in genome D due to Mn-deficiency. The TaNRAMP1 was located in the plasma membrane and showed six motifs matched to Nramp (divalent metal transport). Further, TaNRAMP1 showed a close partnership with cation transporter associated with P-type ATPase/cation transport network. In the RNASeq platform, TaNRAMP1, located in all three genomes, showed the highest expression potential in microspore. Besides, only TaNRAMP1 in genome D was upregulated due to heat and drought stress but showed downregulation in response to excess sulfur and Puccinia triticina infection in all three genomes. The cis-regulatory analysis implies the transcriptional regulation of TaNRAMP1 linked to methyl jasmonate and abscisic acid synthesis. Furthermore, TaNRAMP1 proteins showed similar physicochemical properties, but the C-terminus position of genome D was different than genome A and B. This is the first study on the responses and genome-wide characterization of TaNRAMP1 in Mn-starved wheat.

A novel cysteine-rich receptor-like kinase gene, TaCRK2, contributes to leaf rust resistance in wheat.

Leaf rust, caused by Puccinia triticina, is one of the most destructive fungal diseases in wheat production worldwide. The hypersensitive reaction (HR) is an important defence response against P. triticina infection. In this study, the physiological races 165 and 260 of P. triticina were combined with a line derived from the bread wheat cultivar Thatcher with the leaf rust resistance locus Lr26 to form compatible and incompatible combinations, respectively. Based on an RNA‐Seq database of the interaction systems, a new wheat cysteine‐rich receptor‐like kinase gene, TaCRK2, is specifically induced and up‐regulated in the incompatible combination. We identified that TaCRK2 was regulated in a Ca2+‐dependent manner. Knockdown of TaCRK2 by virus‐induced gene silencing and RNAi leads to a dramatic increase in HR area and the number of haustorial mother cells at the single infection site. In addition, urediniospores, a P. triticina‐specific pathogenic marker in compatible combinations, were observed on leaf surfaces of silenced plants at approximately 15 days after inoculation in the incompatible combination. Moreover, transcription levels of TaPR1, TaPR2, and TaPR5 were obviously reduced in TaCRK2‐silenced plants. TaCRK2 overexpression in Nicotiana benthamiana induced strong HR‐like cell death. Finally, transient expression of green fluorescent protein fused with TaCRK2 in N. benthamiana indicated that TaCRK2 localizes in the endoplasmic reticulum. Thus, TaCRK2 plays an important role in the resistance to P. triticina infection and has a positive regulation effect on the HR cell death process induced by P. triticina.

TaCAMTA4, a Calmodulin-Interacting Protein, Involved in Defense Response of Wheat to Puccinia triticina

Leaf rust caused by Puccinia triticina is one of the main diseases affecting wheat (Triticum aestivum) production worldwide. Calmodulin (CaM) was found involved in the early stage of signal transduction pathway in response to P. triticina in wheat. To study the function and molecular mechanism of calmodulin (CaM) in signal transduction of wheat against P. triticina, we cloned a putative calmodulin-binding transcription activator (TaCAMTA4), and characterized its molecular structure and functions by using the CaM-encoding gene (TaCaM4-1) as a bait to screen the cDNA library from P. triticina infected wheat leaves. The open reading frame of TaCAMTA4 was 2505 bp encoding a protein of 834 aa, which contained all the four conserved domains of family (CG-1 domain, TIG domain, ANK repeats and CaM-binding domain). TaCaM4-1 bound to TaCAMTA4 by the C-terminal CaM-binding domain in Ca2+-dependent manner in the electrophoretic mobility shift assay (EMSA). Bimolecular fluorescence complementation (BiFC) analysis indicated that the interaction of TaCAMTA4 and TaCaM4-1 took place in the cytoplasm and nucleus of epidermal leaf cells in N. benthamiana. The expression level of TaCAMTA4 genes was down-regulated in incompatible combination after P. triticina infection. Furthermore, virus-induced gene silencing (VIGS)-based knockdown of TaCAMTA4 and disease assays verified that silencing of TaCAMTA4 resulted in enhanced resistance to P. triticina race 165. These results suggested that TaCAMTA4 function as negative regulator of defense response against P. triticina.

Functional characterization of the Wrab17 gene in the interaction process between wheat and Puccinia triticina

In the interaction between wheat and Puccinia triticina, wheat resists the invasion of P. triticina by producing hypersensitive reaction-programmed cell death (HR-PCD). To better understand the mechanism of wheat resistance to P. triticina, it is important to identify the defensive genes involved in wheat resistance to leaf rust. This paper systematically presented the role of Wrab17 in the HR-PCD process in wheat after infection by P. triticina. The subcellular location analysis is performed using the full length of Wrab17 coding sequences and Wrab17 is found to be localized in cell nucleus and cytoplasm. Quantitative RT-PCR (RT-qPCR) and western blot analysis showed that expression of Wrab17 at both mRNA and protein levels increased by P. triticina infection, indicating that the Wrab17 gene participated in the interaction between wheat and P. triticina. Wrab17 knockdown plants were generated by RNA-mediated gene silencing technology (RNA interference, RNAi) and confirmed by southern blot. Further analysis with P. triticina inoculation found that knockdown of Wrab17 exhibited decreased HR expansion area in incompatible combination (L10×260) and significant higher sensitivity to the compatible pathogen P. triticina race 165. In all, this work reports that Wrab17 is a new defensive gene playing a role in wheat resistance to leaf rust.

Resistance of (Aegilops tauschii × Secale cereale) × Triticosecale Hybrids to Leaf Rust (Puccinia triticina) Determined on the Macroscopic and Microscopic Level.

Leaf rust caused by Puccinia triticina Eriks belongs to the most important fungal pathogens of wheat (Triticum aestivum L.) and triticale (× Triticosecale). Effective resistance to leaf rust is both, cost-effective and environmentally safe. Many wild Aegilops species carry unknown resistances against fungal diseases and are characterized by a high genetic variability. The main goal of this work was to examine the resistance of (Aegilops tauschii × Secale cereale) × Triticosecale hybrids to leaf rust in inoculation tests with different races of P. triticina. Hybrid plants were selected for the presence of 2D chromosome/s in the triticale background using fluorescence and genomic in situ hybridization. The presence of leaf rust resistance genes was confirmed with closely linked molecular markers, i.e., Xgdm35 and Xgwm296. 14 genotypes of BC2F4 – BC2F6 hybrid plants with the monosomic addition of chromosome 2D (M2DA) were analyzed together with nine control lines. Resistance was determined at the macroscopic and microscopic level at the seedling and adult plant stage (flag leaf). In general, results revealed limited resistance of hybrid plants at the seedling stage, followed by an increase of the resistance level at later stages of plant development. This indicates that respective hybrid plants may exhibit APR resistance conferred by Lr22a introgressed from Ae. tauschii. On the basis of the macroscopic and microscopic analysis, this kind of resistance turned out to be additive and race-specific. We selected four monosomic 2D addition triticale genotypes highly resistant to P. triticina infection at the two main stages of plant development. From the selected genotypes, we obtained 26 doubled haploid lines among which two lines with doubled additional chromosomes 2D of Ae. tauschii can be used for further breeding to increase leaf rust resistance of cultivated triticale.

Changes of Nitric Oxide and Its Relationship with H2O2 and Ca2+ in Defense Interactions between Wheat and Puccinia Triticina.

In this research, the wheat cultivar 'Lovrin 10' and Puccinia triticina races 165 and 260 were used to constitute compatible and incompatible combinations to investigate the relationship between NO and H2O2 and between NO and calcium (Ca2+) signaling in the cell defense process by pharmacological means. The specific fluorescent probe DAF-FM DA was coupled with confocal laser scanning microscopy and used to label intracellular nitric oxide (NO) and monitoring the real-time NO dynamics during the processes of wheat defense response triggered by P. triticina infection. The results showed that at 4 h after inoculation, weak green fluorescence was observed in the stomatal guard cells at the P. triticina infection site in the incompatible combination, which indicates a small amount of NO production. Twelve hours after inoculation, the fluorescence of NO in- cell adjacent to the stomata gradually intensified, and the NO fluorescent area also expanded continuously; the green fluorescence primarily occurred in the cells undergoing a hypersensitive response (HR) at 24–72 h after inoculation. For the compatible combination, however, a small amount of green fluorescence was observed in stomata where the pathogenic contact occurred at 4 h after inoculation, and fluorescence was not observed thereafter. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 h after inoculation and suppressed HR advancement. The injection of imidazole, a NADPH oxidase inhibitor, or EGTA, an extracellular calcium chelator, in the leaves prior to inoculation, delayed the onset of NO production in the incompatible combination and suppressed HR advancement. Combined with our previous results, it could be concluded that, Ca2+ and hydrogen peroxide (H2O2) are involved in upstream of NO production to induce the HR cell death during P. triticina infection, and Ca2+, NO and H2O2 are jointly involved in the signal transduction process of HR in the interaction system.

Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen, Puccinia triticina

TaLr35PR5 gene was obtained from the gDNA and cDNA of TcLr35 wheat. It was induced by Puccinia triticina, ABA and SA, but TaLr35PR5 was induced earlier and its expression level was higher in the incompatible interaction than that in the compatible interaction. In addition, the accumulations of TaLr35PR5 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blottings were conducted to confirm that TaLr35PR5 be induced by P. triticina infection at the protein expression level. Similar to the expression patterns of TaLr35PR5 at RNA levels, the accumulations of TaLr35PR5 protein were weak in the seedling stage, then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene.

Expression and Functional Analysis of a Pathogenesis-Related Protein 1 Gene, TcLr19PR1, Involved in Wheat Resistance Against Leaf Rust Fungus

Pathogenesis-related (PR) proteins encoded by plant defense genes play key roles in plant disease-resistance responses, specialized in systemic-acquired resistance. However, their roles in the response of wheat to fungal infection are still not well known. Our earlier studies have reported that a full-length TcLr19PR1 gene (818 bp) was isolated from wheat infected by leaf rust (Puccinia triticina). Here, we showed that TcLr19PR1 was induced earlier and its expression level was higher in the incompatible interaction between seedling stage of wheat and P. triticina than in compatible interactions. TcLr19PR1 was strongly induced after P. triticina inoculation and ABA and SA treatments, in which the expression level of TcLr19PR1 significantly increased and reached the maximum at 12 and 72 h, respectively. Furthermore, the transgenic T1-stable TcLr19PR1 lines generated in the susceptible wheat cultivar Zhengzhou 5389 background exhibited certain extent disease resistance against P. triticina infection by preventing disease development. In addition, Western blotting was conducted to confirm that TcLr19PR1 protein was induced by P. triticina infection in positive transgenic plants at the protein expression level. These findings suggest that TcLr19PR1 gene plays an important role in wheat development and resistance to leaf rust pathogen attack.

Comparative microscopic and molecular analysis of Thatcher near‐isogenic lines with wheat leaf rust resistance genes Lr2a, Lr3, LrB or Lr9 upon challenge with different Puccinia triticina races

Thatcher near‐isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semiquantitative multiplex PCR analysis using P. triticina‐ and wheat‐specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maximum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P. triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3–4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45·5–51·5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3–4’. Furthermore, a strong deposition of callose was induced in Lr9 + BBBD and Lr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.

Variation of Catechin, epicatechin and their enantiomers concentrations before and after wheat cultivar- Puccinia triticina infection

Three wheat cultivars (Boolani, Roushan and Fornota) were inoculated with Puccinia triticina strain 140 and placed in greenhouse and concentrations of catechin, epicatechin and their enantiomers were monitored in three steps; healthy leaves before and after exposed and also diseased leaves after exposed. The concentration of catechin and epicatechin in infected plant ranged from 4.43 to 11.00 and 1.65 to 3.68 times higher than those in non-infected plant, respectively. Also, the healthy leaves of infected plants had more amounts of catechin and epicatechin than diseased leaves. Study of chiral separations by cellulose column, indicated that both enantiomers of catechin were found in healthy leaves of non-infected plants. However, in the leaves of wheat which were exposed to P. triticina, the concentration of (+) catechin increased while (−)-catechin disappeared in healthy and diseased leaves of infected plants. Furthermore, only (−)-epicatechin was found in all the samples. So, infection by pathogen caused a change in the biosynthesis and accumulation of catechin and also a change in the enantiomeric ration of catechin enantiomers in the leaves.

Preinoculation Effects of Light Quantity on Infection Efficiency of Puccinia striiformis and P. triticina on Wheat Seedlings

ABSTRACT In a previous study under controlled conditions, a model was developed to predict the infection efficiency for the wheat leaf and stripe rust fungi based on temperature and dew period during the 24 h after inoculation. The two pathogens differed in their maximum infection efficiency under controlled conditions for temperature and dew period, the infection efficiency was 12 times greater for Puccinia triticina than for P. striiformis. In the present study, the model was validated by field results to predict P. triticina infection efficiency as a function of temperature and dew period only. However, this model failed to predict infection efficiency caused by P. striiformis in the field. The model was adapted to include the effects of light quantity on infection efficiency. Wheat seedlings, grown in climate-controlled rooms and exposed to various regimes of light duration and intensity for 24 h in either field or controlled conditions, were inoculated and incubated in climate-controlled rooms under optimal dew and temperature conditions. Quantity of natural or artificial light (light intensity x duration) received by the plants prior to inoculation enhanced infection efficiency of wheat seedlings inoculated by P. striiformis. Infection efficiency increased from 0.4 to 36% depending on the light quantity according to a Richards' function. For stripe rust, three environmental variables, preinoculation light quantity received by the plants, postinoculation temperature, and postinoculation dew period, were used for fitting a model for infection efficiency measured in the field.