Flax Rust Fungus Research Articles

Virtual insights into the quercetin- Melampsora lini-derived effector AvrM14 interaction: An In silico exploration of plant defense mechanisms

The co-evolutionary struggle between plants and pathogens shapes vital defense mechanisms crucial for survival. AvrM14 selectively removes the 5′ cap from mRNA, disrupting key aspects of plant mRNA regulation, enhancing the flax rust fungus's infection capabilities. In contrast, quercetin, a plant flavonoid, serves as an antioxidant, stress reliever, and immune protector. This in silico study explores quercetin's inhibition of AvrM14, revealing high-affinity binding to the Nidux domain through molecular docking. Confirming stability in dynamic simulations, this pioneering research establishes quercetin as a functional AvrM14 inhibitor, suggesting its potential as a therapeutic agent against Melampsora lini's harmful impact on plant health.

A rust-fungus Nudix hydrolase effector decaps mRNA invitro and interferes with plant immune pathways.

To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive invitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts invitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage invitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with invitro mRNA-decapping activity capable of interfering with plant immunity.

Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BackgroundRust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field.ResultsTo facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein.ConclusionsThe M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3011-9) contains supplementary material, which is available to authorized users.

Changing the Game: Using Integrative Genomics to Probe Virulence Mechanisms of the Stem Rust Pathogen Puccinia graminis f. sp. tritici.

The recent resurgence of wheat stem rust caused by new virulent races of Puccinia graminis f. sp. tritici (Pgt) poses a threat to food security. These concerns have catalyzed an extensive global effort toward controlling this disease. Substantial research and breeding programs target the identification and introduction of new stem rust resistance (Sr) genes in cultivars for genetic protection against the disease. Such resistance genes typically encode immune receptor proteins that recognize specific components of the pathogen, known as avirulence (Avr) proteins. A significant drawback to deploying cultivars with single Sr genes is that they are often overcome by evolution of the pathogen to escape recognition through alterations in Avr genes. Thus, a key element in achieving durable rust control is the deployment of multiple effective Sr genes in combination, either through conventional breeding or transgenic approaches, to minimize the risk of resistance breakdown. In this situation, evolution of pathogen virulence would require changes in multiple Avr genes in order to bypass recognition. However, choosing the optimal Sr gene combinations to deploy is a challenge that requires detailed knowledge of the pathogen Avr genes with which they interact and the virulence phenotypes of Pgt existing in nature. Identifying specific Avr genes from Pgt will provide screening tools to enhance pathogen virulence monitoring, assess heterozygosity and propensity for mutation in pathogen populations, and confirm individual Sr gene functions in crop varieties carrying multiple effective resistance genes. Toward this goal, much progress has been made in assembling a high quality reference genome sequence for Pgt, as well as a Pan-genome encompassing variation between multiple field isolates with diverse virulence spectra. In turn this has allowed prediction of Pgt effector gene candidates based on known features of Avr genes in other plant pathogens, including the related flax rust fungus. Upregulation of gene expression in haustoria and evidence for diversifying selection are two useful parameters to identify candidate Avr genes. Recently, we have also applied machine learning approaches to agnostically predict candidate effectors. Here, we review progress in stem rust pathogenomics and approaches currently underway to identify Avr genes recognized by wheat Sr genes.

Fungal effector proteins: past, present and future.

The pioneering research of Harold Flor on flax and the flax rust fungus culminated in his gene-for-gene hypothesis. It took nearly 50 years before the first fungal avirulence (Avr) gene in support of his hypothesis was cloned. Initially, fungal Avr genes were identified by reverse genetics and map-based cloning from model organisms, but, currently, the availability of many sequenced fungal genomes allows their cloning from additional fungi by a combination of comparative and functional genomics. It is believed that most Avr genes encode effectors that facilitate virulence by suppressing pathogen-associated molecular pattern-triggered immunity and induce effector-triggered immunity in plants containing cognate resistance proteins. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either on the plasma membrane or inside the plant cell. Indirect recognition of an effector (also known as the guard model) implies that the virulence target of an effector in the host (the guardee) is guarded by the resistance protein (the guard) that senses manipulation of the guardee, leading to activation of effector-triggered immunity. In this article, we review the literature on fungal effectors and some pathogen-associated molecular patterns, including those of some fungi for which no gene-for-gene relationship has been established.

Purification of the M flax-rust resistance protein expressed in Pichia pastoris

The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.

The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells.

The Linum usitatissimum (flax) L gene alleles, which encode nucleotide binding site-Leu rich repeat class intracellular receptor proteins, confer resistance against the Melampsora lini (flax rust) fungus. At least 11 different L resistance specificities are known, and the corresponding avirulence genes in M. lini map to eight independent loci, some of which are complex and encode multiple specificities. We identified an M. lini cDNA marker that cosegregates in an F2 rust family with a complex locus determining avirulence on the L5, L6, and L7 resistance genes. Two related avirulence gene candidates, designated AvrL567-A and AvrL567-B, were identified in a genomic DNA contig from the avirulence allele, whereas the corresponding virulence allele contained a single copy of a related gene, AvrL567-C. Agrobacterium tumefaciens-mediated transient expression of the mature AvrL567-A or AvrL567-B (but not AvrL567-C) proteins as intracellular products in L. usitatissimum and Nicotiana tabacum (tobacco) induced a hypersensitive response-like necrosis that was dependent on coexpression of the L5, L6, or L7 resistance gene. An F1 seedling lethal or stunted growth phenotype also was observed when transgenic L. usitatissimum plants expressing AvrL567-A or AvrL567-B (but not AvrL567-C) were crossed to resistant lines containing L5, L6, or L7. The AvrL567 genes are expressed in rust haustoria and encode 127 amino acid secreted proteins. Intracellular recognition of these rust avirulence proteins implies that they are delivered into host cells across the plant membrane. Differences in the three AvrL567 protein sequences result from diversifying selection, which is consistent with a coevolutionary arms race.

Rust fungi manipulate their host's metabolism

The rusts are a group of obligately biotrophic fungal pathogens that infect only a narrow range of living plant hosts for growth and reproduction. For host colonization, the rusts require the induction of a subset of fungal genes essential for infection. These infection mechanisms are sophisticated, and include the ability to detect the stomate, the entry portal for many rust fungi, and to suppress host-resistance responses. Genes of the plant host are also induced in response to infection, but the role and expression of the host genes have been difficult to dissect because molecular tools such as protocols for transformation and sequenced genomes are lacking for the rusts.Michael Ayliffe et al. [1xA plant gene up-regulated at rust-infection sites. Ayliffe, M.A. et al. Plant Physiol. 2002; 129: 169–180Crossref | PubMed | Scopus (38)See all References[1] have studied the expression of the fis1 gene from flax (Linum usitatissimum) after infection by the flax rust fungus (Melampsora lini). The gene, first identified in 1995 by other members of the group, was induced during a compatible (susceptible) infection but not during an incompatible (resistant) infection. Expression of fis1 was localized exclusively in leaf mesophyll cells closely surrounding the rust infection sites. The data were consistent with the notion that the first hours of a rust infection, before the start of the hypersensitive response in the resistant host, are similar in both the incompatible and compatible interactions, and gene expression was correlated with the amount of fungal growth. Ayliffe et al. [1xA plant gene up-regulated at rust-infection sites. Ayliffe, M.A. et al. Plant Physiol. 2002; 129: 169–180Crossref | PubMed | Scopus (38)See all References[1] also studied the expression of homologous genes isolated from maize (Zea mays, mis1) and barley (Hordeum vulgare, bis1), and showed that these genes, too, were up-regulated by compatible infections with the corresponding species-specific rust. A gene from Arabidopsis (AtP5CDH), highly similar to fis1, encodes Δ1-pyrroline-5-carboxylate dehydrogenase, an enzyme in the pathway that degrades proline to glutamate. Although the authors speculated that this mechanism might provide protection from proline toxicity, other roles are possible, for example, glutamate and glutamine are important amino acids in rust metabolism.The focus of this paper was on the fis1 promoter and its potential for engineering synthetic rust resistance genes, but the paper also demonstrates in a rigorous way that rust fungi can manipulate host metabolism at infection sites. As the authors suggest, there are good reasons to believe that proline metabolism in infected mesophyll cells, where the fungus is located, might be involved either in rust nutrition or in modifying host-defense reactions. It should be possible in the coming years to learn how rusts initiate these changes and determine their roles in host colonization.

Histology of quantitative resistance in flax to the flax rust fungus (Melampsora lini)

Macroscopical and histological research is useful to characterize quantitative resistance. Analysis of histological components of the resistance to the flax rust fungus (Melampsora lini) was carried out on seedlings of seven flax (Linum usitatissimum) accessions: five fibre flax accessions with various levels of quantitative resistance and two near-isogenic lines of linseed with gene K or NI. Flax seedlings were inoculated with uredospores. Collected leaves of the flax accessions were stained with Chlorazol Black E. It was found that in the pathosystem flax - M. lini, a lower number of pustules (lower infection frequency) on quantitatively resistant accessions did not result from mechanisms acting before stomatal penetration by the fungus. Reduced infection frequency was associated with increased rates of early abortion of the rust fungus but rates of this abortion were not large enough to explain the low infection frequency on the quantitatively resistant accessions Afganistan and Solido. In both of the accessions the number of haustoria formed 2 days after inoculation and the size of established colonies after 5 days were reduced. Hyphae of the fungus in the accession Solido stained intensely, and appeared stunted and crinkled. Most of the established colonies in the quantitatively resistant accessions were associated with plant cell necrosis, and at the macroscopic level, pustules were interspersed with some necrotic flecks. This indicates that quantitative resistance of flax to flax rust was caused by incomplete hypersensitivity. Various intensities of the hypersensitive reaction were involved with the resistance of some wild Linum species against M. lini, although in some other accessions it was based on prehaustorial arrestment of the infection units.

Components in the haustorial wall of the flax rust fungus,Melampsora lini, are labelled by three anti-calmodulin monoclonal antibodies

Immunolocalisation studies, using flax leaf material infected with the flax rust fungus,Melampsora lini, and isolated haustorial complexes, have shown that three anti-calmodulin monoclonal antibodies bind to the haustorial wall of the fungus. The epitopes recognised by these antibodies are inserted into the wall during the early stages of haustorium development and remain in the wall throughout the life of the haustorium. The epitopes are present in both compatible and incompatible reactions and are oligosaccharide in nature. The results provide evidence for molecular differentiation within the haustorial complex ofM. lini.

Production and characterisation of monoclonal antibodies to cell wall components of the flax rust fungus

Monoclonal antibodies have been raised against an haustorium-enriched sample prepared from flax leaves infected with the biotrophic flax rust pathogen Melampsora lini. The monoclonal antibodies were produced following conventional and co-immunisation procedures and the range of antibody specificities was compared. The preparation used as immunogen for the conventional protocol was a crude isolate of haustoria consisting of approx. 65% fungal haustoria, the other components being mainly mesophyll cells or cell wall and chloroplast fragments. Following hybridoma production, 40% of positive cell lines produced antibodies that reacted with haustoria and other fungal cells, but 60% bound to plant cells in the infected leaves. For the co-immunisation protocol, the preparation used for immunisation consisted of the crude isolate of haustoria mixed with serum raised against an haustorium-depleted leaf homogenate. In two fusions, 92-94% of the antibodies reacted with fungal cells, including 3 cell lines that localised specifically to the cell wall of haustoria. Only 6-8% of the antibodies produced via co-immunisation reacted with plant cells. The antigens targeted by the three haustorium-specific monoclonal antibodies are incorporated into the wall at early stages of haustorium development, remain in the wall throughout haustorium maturation, and are present in both compatible and incompatible interactions. The epitopes recognised by the monoclonal antibodies are oligosaccharide in nature and the antigens are highly resistant to extraction from the wall. These results highlight the value of the co-immunisation protocol for the production of monoclonal antibodies to specific components in an impure preparation and provide direct evidence for molecular differentiation within the wall of the haustorium of M. lini.

Inhibition of Rust-induced Hypersensitive Response in Flax Cells by the Microtubule Inhibitor Oryzalin

Interactions between the flax rust fungus Melampsora lini and flax Linum usitatissimum L. are governed by a gene-for-gene relationship which determines pathogen virulence or avirulence and host resistance or susceptibility. The present study demonstrates differential sensitivity of M. lini and flax to the microtubule depolymerising drug, oryzalin, such that microtubule depolymerisation in flax cells but not in fungal cells could be obtained. Normally, in an incompatible interaction, a rapid hypersensitive response about 24 h after inoculation inhibits fungal development and invasion. However, in an incompatible interaction in the presence of oryzalin, the occurrence of hypersensitive cell death was delayed and its frequency reduced. This allowed a normally avirulent race of M. lini to form haustoria in living host mesophyll cells at a rate and efficiency similar to that achieved by a virulent race in a compatible interaction during the first 36 h after inoculation. After that time, the incidence of hypersensitive cell death increased and further development of the pathogen was arrested. The results indicate that microtubules play a role in effecting rapid and efficient hypersensitive response in the race–cultivar specific interaction between flax and the flax rust fungus.

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.

New Modes of Genetic Change in Filamentous Fungi

Mendelian genetic analysis of plant pathogenic fungi has been crucial for the discovery of many fundamental principles in plant pathology. The gene-for­ gene hypothesis, for example, an idea essential to understanding host specificity in plant pathogenic fungi, bacteria, and viruses (32), was first articulated by Flor after his exhaustive genetic study of cultivar selectivity in the flax-rust fungus, Melampsora lini (20). More recently, analyses of mei­ otic recombination of traits such as phytoalexin detoxification (99) and mela­ nin biosynthesis (10) in plant pathogenic fungi have confirmed the role of these factors in pathogenicity. However, many important phenomena observed for fungal plant pathogens have never been adequately explained by Mendelian inheritance of pheno­ typic characters. For example, in 1938, Hanson (26) summarized observa­ tions regarding the cultural instability of imperfect plant pathogenic fungi

The inheritance of restriction fragment length polymorphisms in the flax rust Melampsora lini

Random cDNA sequences synthesized from poly A(+) RNA extracted from germinated urediospores of the flax rust fungus, Melampsora lini, were used as probes to detect restriction fragment length polymorphisms (RFLPs) in three races of M. lini originating from cultivated flax, Linum usitatissimum, and one race originating from Australian native flax, L. marginale. Fourteen out of 22 probes tested detected RFLPs in the three races from cultivated flax while 19 of the probes detected polymorphisms between these three races and the race from L. marginale. The segregation of seven RFLPs was determined in a family of 19 F2 progeny derived from a cross between two of the rust races. With six of these the inheritance was consistent, in each case, with the segregation of alleles at a single locus. Inheritance of the seventh was unusual and an explanation involving two loci with null alleles at each was proposed. No linkage was detected between any of the RFLP loci and nine unlinked loci specifying avirulence.

The effects of heat shock and inoculum density on growth and sporulation in axenic cultures of the flax rust fungus

The number of mycelial colonies of Melampsora lini (Ehrenb.) Lév. was increased by a 2-h heat shock at 31 °C delivered 3 h after uredospores were seeded on a liquid medium at 17 ± 0.5 °C under axenic conditions. At 35–38 days from seeding, the ratio of the respective number of colonies in shock and nonshock treatments in 125-mL culture flasks was highest (7.5 to 22) at inoculum levels (1 to 4 × 103 uredospores cm−2) that yielded about 10 colonies per flask without heat shock; the ratio declined sharply at inoculum levels adjusted to yield either smaller or larger numbers of colonies per flask without heat shock. Sporulation (extinction at 458 nm) and protein per colony were positively correlated; were not directly affected by heat shock; were determined by colony number; increased to maxima as mean colony number per flask increased from 1 or less to from 10 to 100 in different experiments; and decreased at higher colony numbers per flask. The results indicate that heat shock promotes the initiation of colonies; that colony number determines the duration of logarithmic growth; and that increasing competition among individual colonies is the primary factor limiting growth and sporulation, whether or not the uredospore germlings are subjected to heat shock.

Further observations on the growth of flax rust fungus in axenic culture

Axenic cultures of flax rust (Melampsora lini (Ehrenb.) Lév.) grown from sterile uredospores on a defined liquid medium in 125-mL Erlenmeyer flasks exhibited an incubation period of 12 to 20 days followed by a period of rapid growth and sporulation. A heat shock (31–33 °C for 2–2.5 h) administered 3 h after seeding the medium did not induce the formation of infection structures, but doubled the number of vegetative colonies initiated and increased total growth per flask (protein/flask) in 4 weeks by 20-fold. The optimum timing of the heat shock corresponded closely with the maximum rate of germ tube emergence. Ambient CO2 was not essential for germination or perception of the heat shock, but was essential, during the first half of the incubation period, for the subsequent development of vegetative colonies. CO2 fixation occurred at all stages of growth from 1 day onward, 14CO2 being incorporated into ethanol-insoluble and ethanol-soluble fractions, including amino acids. Growth rate following the incubation period was not affected by the initial pH of the medium between 5.0 and 6.0, but the incubation period was shorter at pH 6.0.

CO2 is essential for colony initiation by flax rust fungus grown in vitro

Cultures were grown under a constant stream of gas of known composition to eliminate accumulation of any volatiles produced by the fungus. CO2-free air completely inhibits colony initiation. Air containing 1% CO2 allows colony initiation to the same extent as occurs in plugged, nonaerated controls, while "normal air" (ca. 0.03% CO2) causes initiation to be delayed to a variable extent.

Interactions Between Genes Controlling Pathogenicity in the Flax Rust Fungus

Interactions Between Genes Controlling Pathogenicity in the Flax Rust Fungus