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.

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.

Syncytium Induced by Plant-Parasitic Nematodes.

Plant-parasitic nematodes from the genera Globodera, Heterodera (cyst-forming nematodes), and Meloidogyne (root-knot nematodes) are notorious and serious pests of crops. They cause tremendous economic losses between US $80 and 358 billion a year. Nematodes infect the roots of plants and induce the formation of specialised feeding structures (syncytium and giant cells, respectively) that nourish juveniles and adults of the nematodes. The specialised secretory glands enable nematodes to synthesise and secrete effectors that facilitate migration through root tissues and alter the morphogenetic programme of host cells. The formation of feeding sites is associated with the suppression of plant defence responses and deep reprogramming of the development and metabolism of plant cells.In this chapter, we focus on syncytia induced by the sedentary cyst-forming nematodes and provide an overview of ultrastructural changes that occur in the host roots during syncytium formation in conjunction with the most important molecular changes during compatible and incompatible plant responses to infection with nematodes.

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.

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress.

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.

For cells to survive they need to be able to remove faulty or damaged components. The ability to recycle faulty parts is so crucial that some of the molecular machinery responsible is the same across the plant and animal kingdoms. One of the major recycling pathways cells use is autophagy, which labels damaged proteins with molecular tags that say 'eat-me'. Proteins called receptors then recognize these tags and move the faulty component into vesicles that transport the cargo to a specialized compartment that recycles broken parts. Cells make and fold around 40% of their proteins at a site called the endoplasmic reticulum, or ER for short. However, the process of folding and synthesizing proteins is prone to errors. For example, when a cell is under stress this can cause a 'stall' in production, creating a build-up of faulty, partially constructed proteins that are toxic to the cell. There are several quality control systems which help recognize and correct these errors in production. Yet, it remained unclear how autophagy and these quality control mechanisms are linked together. Here, Stephani, Picchianti et al. screened for receptors that regulate the recycling of faulty proteins by binding to the 'eat-me' tags. This led to the identification of a protein called C53, which is found in both plant and animal cells. Microscopy and protein-protein interaction tests showed that C53 moves into transport vesicles when the ER is under stress and faulty proteins start to build-up. Once there, C53 interacts with two proteins embedded in the wall of the endoplasmic reticulum. These proteins form part of the quality control system that senses stalled protein production, labelling the stuck proteins with 'eat-me' tags. Together with C53, they identify and remove half-finished proteins before they can harm the cell. The fact that C53 works in the same way in both plant and human cells suggests that many species might use this receptor to recycle stalled proteins. This has implications for a wide range of research areas, from agriculture to human health. A better understanding of C53 could be beneficial for developing stress-resilient crops. It could also aid research into human diseases, such as cancer and viral infections, that have been linked to C53 and its associated proteins.

Arabidopsis tonoplast intrinsic protein and vacuolar H+-adenosinetriphosphatase reflect vacuole dynamics during development of syncytia induced by the beet cyst nematode Heterodera schachtii.

Plant parasitic cyst nematodes induce specific hypermetabolic syncytial nurse cell structures in host roots. A characteristic feature of syncytia is the lack of the central vacuole and the formation of numerous small and larger vesicles. We show that these structures are formed de novo via widening of ER cisternae during the entire development of syncytium, whereas in advanced stages of syncytium development, larger vacuoles are also formed via fusion of vesicles/tubules surrounding organelle-free pre-vacuole regions. Immunogold transmission electron microscopy of syncytia localised the vacuolar markers E subunit of vacuolar H+-adenosinetriphosphatase (V-ATPase) complex and tonoplast intrinsic protein (γ-TIP1;1) mostly in membranes surrounding syncytial vesicles, thus indicating that these structures are vacuoles and that some of them have a lytic character. To study the function of syncytial vacuoles, changes in expression of AtVHA-B1, AtVHA-B2 and AtVHA-B3 (coding for isoforms of subunit B of V-ATPase), and TIP1;1 and TIP1;2 (coding for γ-TIP proteins) genes were analysed. RT-qPCR revealed significant downregulation of AtVHA-B2, TIP1;1 and TIP1;2 at the examined stages of syncytium development compared to uninfected roots. Expression of VHA-B1 and VHA-B3 decreased at 3 dpi but reached the level of control at 7 dpi. These results were confirmed for TIP1;1 by monitoring At-γ-TIP-YFP reporter construct expression. Infection test conducted on tip1;1 mutant plants showed formation of larger syncytia and higher numbers of females in comparison to wild-type plants indicating that reduced levels or lack of TIP1;1 protein promote nematode development.

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 Role of Programmed Cell Death Regulator LSD1 in Nematode-Induced Syncytium Formation.

Cyst-forming plant-parasitic nematodes are common pests of many crops. They inject secretions into host cells to induce the developmental and metabolic reprogramming that leads to the formation of a syncytium, which is the sole food source for growing nematodes. As in other host-parasite models, avirulence leads to rapid and local programmed cell death (PCD) known as the hypersensitive response (HR), whereas in the case of virulence, PCD is still observed but is limited to only some cells. Several regulators of PCD were analyzed to understand the role of PCD in compatible plant-nematode interactions. Thus, Arabidopsis plants carrying recessive mutations in LESION SIMULATING DISEASE1 (LSD1) family genes were subjected to nematode infection assays with juveniles of Heterodera schachtii. LSD1 is a negative and conditional regulator of PCD, and fewer and smaller syncytia were induced in the roots of lsd1 mutants than in wild-type Col-0 plants. Mutation in LSD ONE LIKE2 (LOL2) revealed a pattern of susceptibility to H. schachtii antagonistic to lsd1. Syncytia induced on lsd1 roots compared to Col0 showed significantly retarded growth, modified cell wall structure, increased vesiculation, and some myelin-like bodies present at 7 and 12 days post-infection. To place these data in a wider context, RNA-sequencing analysis of infected and uninfected roots was conducted. During nematode infection, the number of transcripts with changed expression in lsd1 was approximately three times smaller than in wild-type plants (1440 vs. 4206 differentially expressed genes, respectively). LSD1-dependent PCD in roots is thus a highly regulated process in compatible plant-nematode interactions. Two genes identified in this analysis, coding for AUTOPHAGY-RELATED PROTEIN 8F and 8H were down-regulated in syncytia in the presence of LSD1 and showed an increased susceptibility to nematode infection contrasting with lsd1 phenotype. Our data indicate that molecular regulators belonging to the LSD1 family play an important role in precise balancing of diverse PCD players during syncytium development required for successful nematode parasitism.

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.