Isolation of Natural Fungal Pathogens from Marchantia polymorpha Reveals Antagonism between Salicylic Acid and Jasmonate during Liverwort-Fungus Interactions.

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

Evolution of an Eleusine-Specific Subgroup of Pyricularia oryzae Through a Gain of an Avirulence Gene.

Eleusine isolates (members of the Eleusine pathotype) of Pyricularia oryzae are divided into two subgroups, EC-I and EC-II, differentiated by molecular markers. A multilocus phylogenetic analysis revealed that these subgroups are very close to Eragrostis isolates. EC-II and Eragrostis isolates were exclusively virulent on finger millet and weeping lovegrass, respectively, while EC-I isolates were virulent on both. The avirulence of EC-II on weeping lovegrass was conditioned by an avirulence gene, PWL1. All EC-II isolates shared a peculiar structure (P structure) that was considered to be produced by an insertion (or translocation) of a DNA fragment carrying PWL1. On the other hand, all EC-I and Eragrostis isolates were noncarriers of PWL1 and shared a gene structure that should have predated the insertion of the PWL1-containing fragment. These results, together with phylogenetic analyses using whole-genome sequences, suggest that the Eleusine-specific subgroup (EC-II) evolved through a loss of pathogenicity on weeping lovegrass caused by a gain of PWL1.

The GYF domain protein PSIG1 dampens the induction of cell death during plant-pathogen interactions.

The induction of rapid cell death is an effective strategy for plants to restrict biotrophic and hemi-biotrophic pathogens at the infection site. However, activation of cell death comes at a high cost, as dead cells will no longer be available for defense responses nor general metabolic processes. In addition, necrotrophic pathogens that thrive on dead tissue, take advantage of cell death-triggering mechanisms. Mechanisms by which plants solve this conundrum remain described. Here, we identify PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1) and show that PSIG1 helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in psig1 mutants is independent of salicylic acid (SA) biosynthesis or reactive oxygen species (ROS) production. Moreover, PSIG1 interacts with SMG7, which plays a role in nonsense-mediated RNA decay (NMD), and the smg7-4 mutant allele mimics the cell death phenotype of the psig1 mutants. Intriguingly, the psig1 mutants display enhanced susceptibility to the hemi-biotrophic bacterial pathogen. These findings point to the existence and importance of the SA- and ROS-independent cell death constraining mechanism as a part of the plant immune system.

Identification of a hidden resistance gene in tetraploid wheat using laboratory strains of Pyricularia oryzae produced by backcrossing.

In the process (BC3F1 generation) of backcrossing an Avena isolate of Pyricularia oryzae with a Triticum isolate, color mutants with white mycelia were obtained. These white mutants lacked virulence on all (31/31) hexaploid and most (28/32) tetraploid wheat lines tested. In a BC4F1 population, white and black cultures segregated in a 1:1 ratio, suggesting that the mutant phenotype is controlled by a single gene. Furthermore, the mycelial color was perfectly linked with avirulence in the BC4F1 population; white cultures were all avirulent on common wheat (Triticum aestivum) 'Norin 4' (N4) whereas black cultures were all virulent. White cultures in the BC3F1 and BC4F1 generations were also avirulent on tetraploid wheat (T. dicoccoides) accession 'KU109' (Tat4), which was susceptible to all cultures derived from the parental wild isolates through the BC2F1 generation. A cross between Tat4 and a susceptible tetraploid (T. paleocolchicum) accession 'KU196' (Tat14) produced resistant and susceptible F2 seedlings in a 3:1 ratio against the white cultures. In the F3 generation homozygous resistant/segregating/homozygous susceptible lines segregated in a 1:2:1 ratio. These results suggest that the resistance of Tat4 to the white cultures is controlled by a single major gene. This gene, tentatively designated as RmgTd(t), is considered to be a hidden resistance gene because it was not detected with the Br58, F1, BC1F1, or BC2F1 cultures. Cytological analysis revealed that the moderate resistance controlled by RmgTd(t) was associated with a hypersensitive reaction of mesophyll cells.

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific, toxin-producing fungus Alternaria alternata†Japanese pear pathotype.

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant-pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.

Identification of a novel locus Rmo2 conditioning resistance in barley to host-specific subgroups of Magnaporthe oryzae.

Barley cultivars show various patterns of resistance against isolates of Magnaporthe oryzae and M. grisea. Genetic mechanisms of the resistance of five representative barley cultivars were examined using a highly susceptible barley cultivar, 'Nigrate', as a common parent of genetic crosses. The resistance of the five cultivars against Setaria, Oryza, Eleusine, and Triticum isolates of M. oryzae was all attributed to a single locus, designated as Rmo2. Nevertheless, the Rmo2 locus in each cultivar was effective against a different range of isolates. Genetic analyses of pathogenicity suggested that each cultivar carries an allele at the Rmo2 locus that recognizes a different range of avirulence genes. One allele, Rmo2.a, corresponded to PWT1, which conditioned the avirulence of Setaria and Oryza isolates on wheat, in a gene-for-gene manner. The other alleles, Rmo2.b, Rmo2.c, and Rmo2.d, corresponded to more than one avirulence gene. On the other hand, the resistance of those cultivars to another species, M. grisea, was conditioned by another locus, designated as Rmo3. These results suggest that Rmo2 is effective against a broad range of blast isolates but is specific to M. oryzae. Molecular mapping revealed that Rmo2 is located on the 7H chromosome.

Inhibitory effects of antioxidant reagent in reactive oxygen species generation and penetration of appressoria of Alternaria alternata Japanese pear pathotype.

In the Japanese pear pathotype of Alternaria alternata, H2O2 is generated solely from penetration pegs and not from other portions of subcuticular hyphae within the pectin layers of host leaves. A close association between H2O2 generation and fungal aggressiveness is expected because the pegs are important for fungal penetration into the host epidermis. To determine the potential role of reactive oxygen species in microbial pathogenicity, we studied the inhibitory effects of the antioxidant reagent ascorbic acid and the NADPH oxidase inhibitor diphenylene iodonium on infection of the pathogen. In our study, we showed H2O2 generation to be inhibited by inoculation with the mixture of ascorbic acid or diphenylene iodonium and spores at the pegs in the spore-inoculated host leaves. The decrease of generation in the pegs resulted in penetration failure, indicating that H2O2 generation probably contributed to strengthening of the penetration and probably was recruited by NADPH oxidase.

Evolution of the Eleusine subgroup of Pyricularia oryzae inferred from rearrangement at the Pwl1 locus.

Eleusine isolates (members of the Eleusine subgroup) of Pyricularia oryzae are divided into two groups, Ec-I and Ec-II, differentiated by molecular markers. A multilocus phylogenetic analysis and DNA fingerprinting suggested that Ec-I isolates are very close to Eragrostis isolates rather than Ec-II isolates. Infection assays revealed that Ec-II and Eragrostis isolates were exclusively virulent on finger millet and weeping lovegrass, respectively, whereas Ec-I isolates were virulent on both. The avirulence or virulence on weeping lovegrass perfectly corresponded to the presence or absence of an avirulence gene, PWL1; all Ec-II isolates carried an identical, functional PWL1, whereas none of Ec-I isolates or Eragrostis isolates carried it. A comparison of PWL1 flanking regions revealed that Ec-II isolates had a peculiar structure produced by an insertion (or translocation) of a DNA fragment carrying PWL1. Based on these results, a model was constructed which illustrated possible pathways to the establishment of the Eleusine subgroup.

NoxA activation by the small GTPase RacA is required to maintain a mutualistic symbiotic association between Epichloë festucae and perennial ryegrass.

Small GTPases of the Rac group play a key regulatory role in NADPH oxidase catalysed production of reactive oxygen species (ROS) in mammals and plants, but very little evidence is available for a corresponding role in fungi. We recently showed that ROS produced by a specific fungal NADPH oxidase isoform, NoxA, are crucial in regulating hyphal morphogenesis and growth in the mutualistic symbiotic interaction between Epichloë festucae and perennial ryegrass. We demonstrate here that E. festucae RacA is required for NoxA activation and regulated production of ROS to maintain a symbiotic interaction. Deletion of racA resulted in decreased ROS production, reduction of radial growth and hyper-branching of the hyphae in culture. In contrast, in planta the racA mutant showed extensive colonization of the host plant, resulting in stunting and precocious senescence of the host plants. Strains expressing a dominant active (DA) allele of RacA had increased ROS production, increased aerial hyphae and reduced radial growth. These results demonstrate that RacA plays a crucial role in regulating ROS production by NoxA, in order to control hyphal morphogenesis and growth of the endophyte in planta.