Comparative Genomic Analysis of Effector Repertoires in Rust Fungi: Insights into Pathogenesis and Host Interactions in Wheat

Pucciniales (rust fungi) are one of the largest fungal order of plant pathogens. They collectively infect key crops such as wheat and soybean, and threaten global food security. In the early 2010s, the genome sequences of three rust fungi were released: Melampsora larici-populina (the poplar leaf rust fungus), Puccinia graminis f. sp. tritici (the wheat stem rust fungus), and Puccinia striiformis f. sp. triciti (the wheat stripe rust or wheat yellow rust fungus). The availability of those genomes has forwarded rust biology into the post-genomic era, sparking a series of genomics, transcriptomics, in silico, and functional studies. Here, we snapshot the last 10 years of post-genomics studies addressing M. larici-populina, P. graminis f. sp. tritici, and/or P. striiformis f. sp. tritici. This mini-review notably reveals the model species-centered structure of the research community, and highlights the drastic increase of the number of functional studies focused on effectors since 2014, which notably revealed chloroplasts as a central host compartment targeted by rust fungi. This mini-review also discusses genomics-facilitated studies in other rust species, and emerging post-genomic research trends related to fully-phased rust genomes.

The poplar leaf rust fungus, Melampsora larici-populina has been established as a tree-microbe interaction model. Understanding the molecular mechanisms controlling infection by pathogens appears essential for durable management of tree plantations. In biotrophic plant-parasites, effectors are known to condition host cell colonization. Thus, investigation of candidate secreted effector proteins (CSEPs) is a major goal in the poplar–poplar rust interaction. Unlike oomycetes, fungal effectors do not share conserved motifs and candidate prediction relies on a set of a priori criteria established from reported bona fide effectors. Secretome prediction, genome-wide analysis of gene families and transcriptomics of M. larici-populina have led to catalogs of more than a thousand secreted proteins. Automatized effector-mining pipelines hold great promise for rapid and systematic identification and prioritization of CSEPs for functional characterization. In this review, we report on and discuss the current status of the poplar rust fungus secretome and prediction of candidate effectors from this species.

In the years 1999-2001 field trials were run on susceptibility of wheat and triticale genotypes to infection by three rust fungi (<i>Puccinia recondita, Puccinia graminis, Puccinia striiformis</i>). The results of the observation of the infection level in following years have been similar. Among genotypes of winter wheat, breeding lines susceptible to <i>Puccinia striiformis</i> infection were rare, but among spring wheat 50% of genotypes were susceptible to yellow rust infection. A much higher level of sensitivity than in the case of winter wheat has been found in winter triticale genotypes. Wheat genotypes were distinguished by the high sensitivity to <i>Puccinia graminis</i> infection, only a few breeding lines were resistant to stem rust. The susceptibility of wheat to brown rust (<i>Puccinia recondita</i>) was a common feature. Triticale genotypes compared to wheat were affected significantly less and majority of them exhibited high level of resistant to brown rust. The use of the breeding method has justification in control yellow rust of winter wheat. Recommended cultivars are almost all fully resistant to <i>Puccinia striiformis</i> infection. The application of this method in selection of spring wheat and triticale is in large past limited. Some of the registered cultivars of spring wheat and triticale are very susceptible to yellow rust. Using the breeding method to protect wheat from stem rust and brown rust is of little practical benefit in our county at this moment. But it can be effecive to control stem and brown rusts of triticale.

Ethiopia is the largest producer of wheat (Triticum aestivum L.) in the sub-Saharan Africa. Despite its importance as food and industrial crop, wheat production and productivity around the globe is hampered by a number of factors including biotic and a biotic stresses as well as low adoption of new agricultural technologies. Of the biotic stresses, diseases caused by fungi are the most important factors constraining wheat production. Among the diseases stem rust and yellow rust, are highly destructive diseases of wheat worldwide. The recurrent occurrence of yellow rust and stem rust, diseases in the study area causes considerable yield losses. To prevent the yield loss due to these diseases, farmers used different fungicides released for either wheat diseases or other crop diseases alone or in a combination of fungicides with each other due to the disease's aggressiveness under field conditions. Field experiment was conducted to verify the effectiveness of the Wabe300 EC fungicide (Tebuconazole 225g/L + Triadimenol 75g/L) relative to another promising standard fungicide, Royal 750 WDG, for the management of stem rust and yellow rust of wheat. The experiment was conducted at Kokate, Dalibo Wogane and Zala Shasha, on farmers’ field in a Randomized Complete Block Design (RCBD) with five replications during 2023 cropping season. The experiment result confirmed that, fungicide treated plots were showed significant difference compared to the control treatments in all variables. Wabe 300 EC foliar application at the rate of 0.5 L/ha with 200 liter water per hectare was highly effective in controlling yellow and stem rust of wheat and increased grain yield of wheat. Hence, it is recommended for registration for the management of stem and yellow rust in wheat production. During the growing periods, no foliar toxic effect was observed from the effect of any tested fungicides.

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N.benthamiana. The use of N.benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

Cereal powdery mildews caused by Blumeria graminis and cereal rusts caused by Puccinia spp. are constant disease threats that limit the production of almost all important cereal crops. Rice is an intensively grown agricultural cereal that is atypical because of its immunity to all powdery mildew and rust fungi. We analyzed the nonhost interactions between rice and the wheat powdery mildew fungus B. graminis f. sp. tritici (Bgt) and the wheat leaf rust fungus Puccinia triticina (Ptr) to identify the basis of nonhost resistance (NHR) in rice against cereal powdery mildew and rust fungi at cytological and molecular levels. No visible symptoms were observed on rice leaves inoculated with Bgt or Ptr. Microscopic observations showed that both pathogens exhibited aberrant differentiation and significantly reduced penetration frequencies on rice compared to wheat. The development of Bgt and Ptr was also completely arrested at early infection stages in cases of successful penetration into rice leaves. Attempted infection of rice by Bgt and Ptr induced similar defense responses, including callose deposition, accumulation of reactive oxygen species, and hypersensitive response in rice epidermal and mesophyll cells, respectively. Furthermore, a set of defense-related genes were upregulated in rice against Bgt and Ptr infection. Rice is an excellent monocot model for genetic and molecular studies. Therefore, our results demonstrate that rice is a useful model to study the mechanisms of NHR to cereal powdery mildew and rust fungi, which provides useful information for the development of novel and durable strategies to control these important pathogens.

Cereal rusts, caused by obligate and biotrophic fungi in the genus Puccinia, are important diseases that threaten world food security. With the recent discovery of alternate hosts for the stripe rust fungus (Puccinia striiformis), all cereal rust fungi are now known to be heteroecious, requiring two distinct plant species serving as primary or alternate hosts to complete their sexual life cycle. The roles of the alternate hosts in disease epidemiology and pathogen variation vary greatly from species to species and from region to region because of different climatic and cropping conditions. We focus this review on rust fungi of small grains, mainly stripe rust, stem rust, leaf rust, and crown rust of wheat, barley, oat, rye, and triticale, with emphases on the contributions of alternate hosts to the development and management of rust diseases.

In an effort to enhance wheat's resilience against rust diseases, our research explores the genetic underpinnings of resistance in a diverse collection of winter bread wheat accessions. Leaf rust (Puccinia triticina), yellow rust (Puccinia striiformis f. sp. tritici), and stem rust (Puccinia graminis f. sp. tritici) are significant threats to global wheat production. By leveraging host genetic resistance, we can improve disease management strategies. Our study evaluated 55 wheat accessions, including germplasm from Kazakhstan, from Uzbekistan, from Russia, from Kyrgyzstan, France, and CIMMYT under field conditions in southern Kazakhstan from 2022 to 2024. The results showed a robust resistance profile: 49.1% of accessions exhibited high to moderate resistance to leaf rust, 12.7% to yellow rust, and 30.9% to stem rust. Notably, ten accessions demonstrated resistance to multiple rust species, while seven showed resistance to two rusts. Twenty accessions were selected for further seedling resistance and molecular analysis. Three accessions proved resistant to six isolates of P. triticina, two to four isolates of P. striiformis, and four to five isolates of P. graminis. Although no genotypes were found to be universally resistant to all rust species at the seedling stage, two accessions-Bezostaya 100 (Russia) and KIZ 90 (Kazakhstan)-displayed consistent resistance to leaf and stem rust in both seedling and field evaluations. Molecular analysis revealed the presence of key resistance genes, including Lr1, Lr3, Lr26, Lr34, Yr9, Yr18, Sr31, Sr57, and the 1AL.1RS translocation. This work provides valuable insights into the genetic landscape of wheat rust resistance and contributes to the development of new wheat cultivars that can withstand these diseases, enhancing global food security.

Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.

Common winter wheat is the main grain crop cultivated in the North Caucasus. Rust disease damage isone of the factors limiting wheat productivity. There are three species of rust in the region: leaf (Puccinia triticina),stem (P. graminis) and stripe rust (P. striiformis), and their signif icance varies from year to year. The most commonis leaf rust, but in the last decade the frequency of its epiphytotic development has signif icantly decreased. At thesame time, an increase in the harmfulness of stripe rust (P. striiformis) is noted. Stem rust in the region is mainlyabsent or observed at the end of the wheat growing season to a weak degree. Only in some years with favorableweather conditions its mass development is noted on susceptible cultivars. It is believed that the sources of infectionwith rust species in the North Caucasus are infested soft wheat crops, wild-growing cereals and exodemicinfection carried by air currents from adjacent territories. In the North Caucasus, forage and wild grasses areaffected by Puccinia species almost every year. Depending on weather conditions, the symptom expression isnoted from late September to December and then from late February to May–June. Potentially, an autumn infectionon grasses can serve as a source for infection of winter soft wheat cultivars sown in October. The purpose ofthese studies is to characterize the virulence of P. triticina, P. graminis, P. striiformis on wild cereals and to assessthe specialization of causative agents to winter wheat in the North Caucasus. Infectious material representedby leaves with urediniopustules of leaf, stem and stripe rusts was collected from wild cereals (Poa spp., Bromusspp.) in the Krasnodar Territory in October–November 2019. Uredinium material from P. triticina, P. striiformis, andP. graminis was propagated and cloned. Monopustular Puccinia spp. isolates were used for virulence geneticsanalysis. In experiments to study the specialization of rust species from wild-growing cereals on common wheat,12 winter cultivars were used (Grom, Tanya, Yuka, Tabor, Bezostaya 100, Yubileynaya 100, Vekha, Vassa, Alekseich,Stan, Gurt, Bagrat). These cultivars are widely cultivated in the North Caucasus region and are characterized byvarying degrees of resistance to rust. Additionally, wheat material was inoculated with Krasnodar populations ofP. triticina, P. striiformis, P. graminis from common wheat. In the virulence analysis of P. triticina on cereal grasses,four phenotypes (races) were identif ied: MCTKH (30 %), TCTTR (30 %), TNTTR (25 %), MHTKH (15 %), and f ivewere identif ied in P. graminis (RKMTF (60 %), TKTTF, RKLTF, QKLTF, LHLPF (10 % each). Among P. striiformis isolates,three phenotypes were identif ied using the International and European sets of differentiating cultivars –111E231 (88 %), 111E247 (6 %) and 78E199 (6 %). Using isogenic Avocet lines, 3 races were also identif ied, whichdiffered among themselves in virulence to the Yr1, Yr11, Yr18 genes (with the prevalence of virulent ones (94 %)).Composite urediniums’ samples (a mixture of all identif ied races) of grass rust of each species were used to inoculatewinter wheat cultivars. The most common winter wheat cultivars (75 %) were characterized by a resistantresponse when infected with P. graminis populations from common wheat and cereal grasses. All these cultivarswere developed using donors of the rye translocation 1BL.1RS, in which the Lr26, Sr31, and Yr9 genes are localized.The number of winter wheat cultivars resistant to leaf rust in the seedling phase was lower (58 %). At thesame time, all the studied cultivars in the seedling phase were susceptible to P. striiformis to varying degrees. Thevirulence analysis of the leaf, stem and stripe rust populations did not reveal signif icant differences in the virulenceof the pathogens between wild-growing cereals and soft wheat. Urediniomaterial of all studied rust speciessuccessfully infested soft wheat cultivars. The results obtained indicate that grasses are rust infection reservoirsfor common wheat crops in the North Caucasus.

One of the causative agents of pokkah boeng disease (PBD), which affects sugarcane crops globally, is the fungus Fusarium sacchari. These fungal infections reduce sugar quality and yield, resulting in severe economic losses. Effector proteins play important roles in the interactions between pathogenic fungi and plants. Here, we used bioinformatic prediction approaches to identify 316 candidate secreted effector proteins (CSEPs) in the complete genome of F. sacchari. In total, 95 CSEPs contained known conserved structures, representing 40 superfamilies and 18 domains, while an additional 91 CSEPs contained seven known motifs. Of the 130 CSEPs containing no known domains or motifs, 14 contained one of four novel motifs. A heterogeneous expression system in Nicotiana benthamiana was used to investigate the functions of 163 CSEPs. Seven CSEPs suppressed BAX-triggered programmed cell death in N. benthamiana, while four caused cell death in N. benthamiana. The expression profiles of these eleven CSEPs during F. sacchari infection suggested that they may be involved in sugarcane-F. sacchari interaction. Our results establish a basis for further studies of the role of effector molecules in pathogen–sugarcane interactions, and provide a framework for future predictions of pathogen effector molecules.

Crops are constantly exposed to pathogenic microbes. Rust fungi are examples of these harmful microorganisms, which have a major economic impact on wheat production. To protect themselves from pathogens like rust fungi, plants employ a multilayered immune system that includes immunoreceptors encoded by resistance genes. Significant efforts have led to the isolation of numerous resistance genes against rust fungi in cereals, especially in wheat. However, the evolution of virulence of rust fungi hinders the durability of resistance genes as a strategy for crop protection. Rust fungi, like other biotrophic pathogens, secrete an arsenal of effectors to facilitate infection, and these are the molecules that plant immunoreceptors target for pathogen recognition and mounting defense responses. When recognized, these effector proteins are referred to as avirulence (Avr) effectors. Despite the many predicted effectors in wheat rust fungi, only five Avr genes have been identified, all from wheat stem rust. Knowledge of the Avr genes and their variation in the fungal population will inform deployment of the most appropriate wheat disease-resistance genes for breeding and farming. The review provides an overview of methodologies as well as the validation techniques that have been used to characterize Avr effectors from wheat stem rust. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Pollen analysis identified domestic Zea mays (corn, maize), Phaseolus vulgaris (kidney bean), Helianthus annuus (sunflower) and Cucurbita pepo (squash) accompanied by spores of fungi, many of which cause crop diseases, such as Ustilago maydis (=U. zeae, corn smut), Puccinia sorghi (corn rust), Uromyces appendiculatus (bean rust) and Puccinia graminis (wheat rust). Spores were most abundant in two intervals, ca. a.d. 1300 to ca. 1500 when prehistoric Iroquoian agriculture flourished near the lake and since 1830, when historic Triticum aestivum (wheat) agriculture began. In addition to dispersal by wind, Branta canadensis (Canada goose) also transported fungal spores to the lake via their dung. Spores of fungi that parasitize crop plants may be more abundant than pollen of their host plants and therefore spores may more readily indicate ancient agriculture.

Gel electrophoresis was used to examine the variation in isozymes and dsRNA within and between the rust species Puccinia striiformis, P. recondita and P. hordei. No differences in isozyme phenotype were found among 29 diverse isolates of the wheat‐attacking form of P. striiformis (WYR). Smaller numbers of isolates of the barley‐attacking form (BYR) of P. recondita and of P. hordei showed similar intra‐group uniformity. There were major differences in isozyme phenotypes between the three species, while WYR and BYR differed for two of 12 enzymes. Double‐stranded RNA was detected in each species and in all 26 isolates examined. For WYR and BYR, all isolates within each group had the same or a similar phenotype. In contrast, each isolate of P. recondita and P. hordei had a unique phenotype. There were differences in dsRNA phenotypes both between the three species and between WYR and BYR. The uniformity of these rust populations and species for isozyme phenotype is contrasted with their variability in pathogenicity and with the variability in isozymes encountered in higher organisms. Uniformity may result from a feature of the biology of the rust species and populations examined, or from the relative homogeneity of the environment of biotrophic pathogens. Consistent differences in isozyme and dsRNA phenotype between the WYR and BYR isolates of P. striiformis indicate that these are two discrete populations, supporting the view that they should be recognized as f.sp. tritici and f.sp. hordei , respectively.