Extracellular RNAs released by plant-associated fungi: from fundamental mechanisms to biotechnological applications.

Extracellular RNAs are an emerging research topic in fungal-plant interactions. Fungal plant pathogens and symbionts release small RNAs that enter host cells to manipulate plant physiology and immunity. This communication via extracellular RNAs between fungi and plants is bidirectional. On the one hand, plants release RNAs encapsulated inside extracellular vesicles as a defense response as well as for intercellular and inter-organismal communication. On the other hand, recent reports suggest that also full-length mRNAs are transported within fungal EVs into plants, and these fungal mRNAs might get translated inside host cells. In this review article, we summarize the current views and fundamental concepts of extracellular RNAs released by plant-associated fungi, and we discuss new strategies to apply extracellular RNAs in crop protection against fungal pathogens. KEY POINTS: • Extracellular RNAs are an emerging topic in plant-fungal communication. • Fungi utilize RNAs to manipulate host plants for colonization. • Extracellular RNAs can be engineered to protect plants against fungal pathogens.

Engineering and Implementation of Synthetic Molecular Tools in the Basidiomycete Fungus Ustilago maydis.

The basidiomycete Ustilago maydis is a well-characterized model organism for studying pathogen-host interactions and of great interest for a broad spectrum of biotechnological applications. To facilitate research and enable applications, in this study, three luminescence-based and one enzymatic quantitative reporter were implemented and characterized. Several dual-reporter constructs were generated for ratiometric normalization that can be used as a fast-screening platform for reporter gene expression, applicable to in vitro and in vivo detection. Furthermore, synthetic bidirectional promoters that enable bicisitronic expression for gene expression studies and engineering strategies were constructed and implemented. These noninvasive, quantitative reporters and expression tools will significantly widen the application range of biotechnology in U. maydis and enable the in planta detection of fungal infection.

A mixed-linkage (1,3;1,4)-ß-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide.

The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

How Do Smut Fungi Use Plant Signals to Spatiotemporally Orientate on and In Planta?

Smut fungi represent a large group of biotrophic plant pathogens that cause extensive yield loss and are also model organisms for studying plant-pathogen interactions. In recent years, they have become biotechnological tools. After initial penetration of the plant epidermis, smut fungi grow intra-and intercellularly without disrupting the plant-plasma membrane. Following the colonialization step, teliospores are formed and later released. While some smuts only invade the tissues around the initial penetration site, others colonize in multiple plant organs resulting in spore formation distal from the original infection site. The intimate contact zone between fungal hyphae and the host is termed the biotrophic interaction zone and enables exchange of signals and nutrient uptake. Obviously, all steps of on and in planta growth require fine sensing of host conditions as well as reprogramming of the host by the smut fungus. In this review, we highlight selected examples of smut fungal colonization styles, directional growth in planta, induction of spore formation, and the signals required, pointing to excellent reviews for details, to draw attention to some of the open questions in this important research field.

Genetic Manipulation of the Brassicaceae Smut Fungus Thecaphora thlaspeos.

Investigation of plant-microbe interactions greatly benefit from genetically tractable partners to address, molecularly, the virulence and defense mechanisms. The smut fungus Ustilago maydis is a model pathogen in that sense: efficient homologous recombination and a small genome allow targeted modification. On the host side, maize is limiting with regard to rapid genetic alterations. By contrast, the model plant Arabidopsis thaliana is an excellent model with a vast amount of information and techniques as well as genetic resources. Here, we present a transformation protocol for the Brassicaceae smut fungus Thecaphora thlaspeos. Using the well-established methodology of protoplast transformation, we generated the first reporter strains expressing fluorescent proteins to follow mating. As a proof-of-principle for homologous recombination, we deleted the pheromone receptor pra1. As expected, this mutant cannot mate. Further analysis will contribute to our understanding of the role of mating for infection biology in this novel model fungus. From now on, the genetic manipulation of T. thlaspeos, which is able to colonize the model plant A. thaliana, provides us with a pathosystem in which both partners are genetically amenable to study smut infection biology.

Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors.

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long time scales without macroscopic symptoms. However, how plant-pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown. Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi. The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of these, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection. Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long-lived infection of its perennial hosts.

The Plant-Dependent Life Cycle of Thecaphora thlaspeos: A Smut Fungus Adapted to Brassicaceae.

Smut fungi are globally distributed plant pathogens that infect agriculturally important crop plants such as maize or potato. To date, molecular studies on plant responses to smut fungi are challenging due to the genetic complexity of their host plants. Therefore, we set out to investigate the known smut fungus of Brassicaceae hosts, Thecaphora thlaspeos. T. thlaspeos infects different Brassicaceae plant species throughout Europe, including the perennial model plant Arabis alpina. In contrast to characterized smut fungi, mature and dry T. thlaspeos teliospores germinated only in the presence of a plant signal. An infectious filament emerges from the teliospore, which can proliferate as haploid filamentous cultures. Haploid filaments from opposite mating types mate, similar to sporidia of the model smut fungus Ustilago maydis. Consistently, the a and b mating locus genes are conserved. Infectious filaments can penetrate roots and aerial tissues of host plants, causing systemic colonization along the vasculature. Notably, we could show that T. thlaspeos also infects Arabidopsis thaliana. Exploiting the genetic resources of A. thaliana and Arabis alpina will allow us to characterize plant responses to smut infection in a comparative manner and, thereby, characterize factors for endophytic growth as well as smut fungi virulence in dicot plants.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Fungal chitinases: function, regulation, and potential roles in plant/pathogen interactions.

In the past decades our knowledge about fungal cell wall architecture increased tremendously and led to the identification of many enzymes involved in polysaccharide synthesis and remodeling, which are also of biotechnological interest. Fungal cell walls play an important role in conferring mechanic stability during cell division and polar growth. Additionally, in phytopathogenic fungi the cell wall is the first structure that gets into intimate contact with the host plant. A major constituent of fungal cell walls is chitin, a homopolymer of N-acetylglucosamine units. To ensure plasticity, polymeric chitin needs continuous remodeling which is maintained by chitinolytic enzymes, including lytic polysaccharide monooxygenases N-acetylglucosaminidases, and chitinases. Depending on the species and lifestyle of fungi, there is great variation in the number of encoded chitinases and their function. Chitinases can have housekeeping function in plasticizing the cell wall or can act more specifically during cell separation, nutritional chitin acquisition, or competitive interaction with other fungi. Although chitinase research made huge progress in the last decades, our knowledge about their role in phytopathogenic fungi is still scarce. Recent findings in the dimorphic basidiomycete Ustilago maydis show that chitinases play different physiological functions throughout the life cycle and raise questions about their role during plant-fungus interactions. In this work we summarize these functions, mechanisms of chitinase regulation and their putative role during pathogen/host interactions.

Chitinases Are Essential for Cell Separation in Ustilago maydis.

Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

Microtubule-dependent membrane dynamics in Ustilago maydis: Trafficking and function of Rab5a-positive endosomes.

Long-distance trafficking of membranous structures along the cytoskeleton is crucial for secretion and endocytosis in eukaryotes. Molecular motors are transporting both secretory and endocytic vesicles along polarized microtubules. Here, we review the transport mechanism and biological function of a distinct subset of large vesicles marked by the G-protein Rab5a in the model microorganism Ustilago maydis. These Rab5a-positive endosomes shuttle bi-directionally along microtubules mediated by the Unc104/KIF1A-related motor Kin3 and dynein Dyn1/2. Rab5a-positive endosomes exhibit diverse functions during the life cycle of U. maydis. In haploid budding cells they are involved in cytokinesis and pheromone signaling. During filamentous growth endosomes are used for long-distance transport of mRNA, a prerequisite to maintain polarity most likely via local translation of specific proteins at both the apical and distal ends of filaments. Endosomal co-transport of mRNA constitutes a novel function of these membrane compartments supporting the view that endosomes function as multipurpose platforms.

Molecular crosstalk between PAMP-triggered immunity and photosynthesis.

The innate immune system allows plants to respond to potential pathogens in an appropriate manner while minimizing damage and energy costs. Photosynthesis provides a sustained energy supply and, therefore, has to be integrated into the defense against pathogens. Although changes in photosynthetic activity during infection have been described, a detailed and conclusive characterization is lacking. Here, we addressed whether activation of early defense responses by pathogen-associated molecular patterns (PAMPs) triggers changes in photosynthesis. Using proteomics and chlorophyll fluorescence measurements, we show that activation of defense by PAMPs leads to a rapid decrease in nonphotochemical quenching (NPQ). Conversely, NPQ also influences several responses of PAMP-triggered immunity. In a mutant impaired in NPQ, apoplastic reactive oxygen species production is enhanced and defense gene expression is differentially affected. Although induction of the early defense markers WRKY22 and WRKY29 is enhanced, induction of the late markers PR1 and PR5 is completely abolished. We propose that regulation of NPQ is an intrinsic component of the plant's defense program.

How microbes utilize host ubiquitination.

Activity, abundance and localization of eukaryotic proteins can be regulated through covalent attachment of ubiquitin and ubiquitin-like moieties. Ubiquitination is important in various aspects of immunity. Pathogens utilize host ubiquitination for the suppression of immune signalling and reprogramming host processes to promote microbial life. They deliver so-called effector molecules into host cells, which functionally or structurally resemble components of the host ubiquitination machinery utilizing this enzymatic process or they secrete molecules to inhibit ubiquitin-mediated degradation. Since prokaryotic pathogens lack a classical ubiquitination system, effector mimicry of components of the ubiquitin machinery could be achieved through gene flow. Horizontal gene transfer allows pathogenic bacteria to access ubiquitination enzymes from a potential host, while lateral gene transfer recruits components from another pathogen providing spread within the microbial community. Additionally, convergent evolution can shape bacterial proteins to acquire ubiquitination functions.

Plant pattern-recognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB.

BACKGROUND: An important layer of active defense in plant immunity is the detection of pathogen-associated molecular patterns (PAMPs) mediated by cell-surface receptors. For the establishment of disease, pathogens depend on the ability to overcome PAMP perception and disable plant signaling pathways activated in response to PAMPs. Pattern recognition receptors (PRRs) are therefore prime targets for pathogen effectors. FLS2, its coreceptor BAK1, and EFR encode receptor-like kinases that play a role in immunity against bacterial pathogens. RESULTS: Here, we report that virulence of Pseudomonas syringae pv tomato DC3000 (PtoDC3000) in Arabidopsis is enhanced through the action of its effector AvrPtoB, which promotes degradation of FLS2. We show that AvrPtoB, through its N terminus, associates with FLS2 and BAK1, of which interaction with FLS2 is enhanced by flg22 activation. In vitro, AvrPtoB is active as an E3 ligase to catalyze polyubiquitination of the kinase domain of FLS2, a process confirmed in planta. Full enhancement of PtoDC3000 virulence appears to require the E3 ligase activity of AvrPtoB. CONCLUSIONS: AvrPtoB, initially identified through its activation of hypersensitive resistance in tomato cultivars expressing the Pto kinase, is composed of at least two functional domains: the N terminus is responsible for interaction with Pto, and the C terminus carries an E3 ligase activity. Based on our findings, we propose that both domains of AvrPtoB act together to support the virulence of PtoDC3000 in Arabidopsis through their ability to eliminate FLS2 from the cell periphery, and probably also other PAMP sensors that are constitutively expressed or induced after pathogen challenge.

Breaking the barriers: microbial effector molecules subvert plant immunity.

Adaptation to specialized environments allows microorganisms to inhabit an enormous variety of ecological niches. Growth inside plant tissues is a niche offering a constant nutrient supply, but to access this niche, plant defense mechanisms ranging from passive barriers to induced defense reactions have to be overcome. Pathogens have to break several, if not all, of these barriers. For this purpose, they secrete effector molecules into plant cells to interfere with individual defense responses. Plant defense is organized in multiple layers, and therefore the action of effectors likely follows this same order, leading to a hierarchy in effector orchestration. In this review we summarize the latest findings regarding the level at which effectors manipulate plant immunity. Particular attention is given to those effectors whose mechanism of action is known. Additionally, we compare methods to identify and characterize effector molecules.

One of two alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas.

Proteins of the YidC/Oxa1p/ALB3 family play an important role in inserting proteins into membranes of mitochondria, bacteria, and chloroplasts. In Chlamydomonas reinhardtii, one member of this family, Albino3.1 (Alb3.1), was previously shown to be mainly involved in the assembly of the light-harvesting complex. Here, we show that a second member, Alb3.2, is located in the thylakoid membrane, where it is associated with large molecular weight complexes. Coimmunoprecipitation experiments indicate that Alb3.2 interacts with Alb3.1 and the reaction center polypeptides of photosystem I and II as well as with VIPP1, which is involved in thylakoid formation. Moreover, depletion of Alb3.2 by RNA interference to 25 to 40% of wild-type levels leads to a reduction in photosystems I and II, indicating that the level of Alb3.2 is limiting for the assembly and/or maintenance of these complexes in the thylakoid membrane. Although the levels of several photosynthetic proteins are reduced under these conditions, other proteins are overproduced, such as VIPP1 and the chloroplast chaperone pair Hsp70/Cdj2. These changes are accompanied by a large increase in vacuolar size and, after a prolonged period, by cell death. We conclude that Alb3.2 is required directly or indirectly, through its impact on thylakoid protein biogenesis, for cell survival.

Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation.

High concentrations of heavy metals (HM) in the soil have detrimental effects on ecosystems and are a risk to human health as they can enter the food chain via agricultural products or contaminated drinking water. Phytoremediation, a sustainable and inexpensive technology based on the removal of pollutants from the environment by plants, is becoming an increasingly important objective in plant research. However, as phytoremediation is a slow process, improvement of efficiency and thus increased stabilization or removal of HMs from soils is an important goal. Arbuscular mycorrhizal (AM) fungi provide an attractive system to advance plant-based environmental clean-up. During symbiotic interaction the hyphal network functionally extends the root system of their hosts. Thus, plants in symbiosis with AM fungi have the potential to take up HM from an enlarged soil volume. In this review, we summarize current knowledge about the contribution of the AM symbiosis to phytoremediation of heavy metals.

Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3.

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.