Issei Kobayashi

Preparation of the infection court by Erysiphe graminis: Degradation of the host cuticle☆

Abstract Contact with a surface stimulates the release of a liquid from conidia of Erysiphe graminis and the liquid has been shown to contain esterase activity. The present investigation demonstrates that the liquid exudate partially erodes the cuticle of barley leaves. The release of esterase into the liquid occurs in two stages. The second stage of release occurs between 10 and 15 min after contact and was shown to be temperature dependent and to be inhibited by cycloheximide. Cuticular erosion may provide a stimulus for subsequent morphological differentiation, such as appressorium formation, or it may enhance appressorial adhesion.

Actin-related defense mechanism to reject penetration attempt by a non-pathogen is maintained in tobacco BY-2 cells

The actin cytoskeleton is a key player in defense responses during early stages of infection by fungal pathogens. To investigate molecular mechanisms of actin-related defense responses, a cultured tobacco (Nicotiana tabacum L.) BY-2 cell system was devised. When conidia were directly deposited on BY-2 cells, neither a pathogen, Erysiphe cichoracearum, nor a non-pathogen, Erysiphe pisi, was able to form appressoria or haustoria on BY-2 cells. On the other hand, conidia of the powdery mildews formed appressoria on BY-2 cells if they were covered with a thin hydrophobic membrane of Formvar. Percentages of appressoria formation of the powdery mildews on the Formvar-covered BY-2 cells were mostly the same as those on leaf epidermal cells. The pathogen successfully penetrated through the membrane into BY-2 cells and formed haustoria, whereas penetration attempts of the non-pathogen were completely rejected by the BY-2 cells similar to attempts on leaf epidermal cells. On the other hand, when BY-2 cells were treated with actin cytoskeleton-depolymerizing agents, cytochalasins, the non-pathogen became able to penetrate and form haustoria in BY-2 cells. Simultaneously, cytochalasin inhibited callose deposition at penetration sites of the non-pathogen. These results demonstrated that the actin cytoskeleton plays an important role in defense mechanisms against fungal penetration, even in the dedifferentiated cultured cells. The newly devised Formvar-covered cultured cell system will be a useful tool for molecular dissection of signal perception and defense mechanisms of plant cells during the early stage of fungal attack.

Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.

Depolymerization of the actin cytoskeleton induces defense responses in tobacco plants

Tobacco leaf sections were treated with actin inhibitors, i.e., cytochalasins, to determine the effects of actin depolymerization on tobacco defense responses. Inoculation of the leaf sections with the pathogen Erysiphe cichoracearum, depolymerized the actin cytoskeleton, priming the cells for a hypersensitive response-like cell death. Further, expression of the acidic PR1 and PR2 genes were induced in cytochalasin-treated leaf sections. The intensity of the cytochalasin effects on the defense responses was closely correlated with the extent of actin depolymerization. This suggests that plant cells may perceive perturbation of the actin cytoskeleton, and this stimulus may trigger plant defense responses.

Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. VII. Enhancement of inaccessibility at the prepenetration stage of a nonpathogen.

The inaccessibility enhanced by a nonpathogen, Erysiphe pisi, in coleoptile cells of barley at the prepenetration stage was related to the time of appressorium maturation. Within 4–5 h after inoculation a unique circular to semicircular structure became visible by light microscopy in one of the lobes of the polymorphic appressorium. Micromanipulation at the SEM level revealed that this structure represented the bottom of the lobe appressed to the host surface. This structure was used as an indicator for maturation of the appressorium. To relate the time of appressorial maturation to induction of inaccessibility, germlings of E. pisi were removed from coleoptile cell surfaces with a micromanipulator and germlings of E. graminis were transferred from another coleoptile to the surface of the same cell. Penetration efficiency (the rate of haustorium formation) of E. graminis was suppressed to about 50% of that of controls when E. pisi had been removed at or 2 h after maturation of its appressorium, while it was not affected when E. pisi had been removed before maturation of its appressorium. These results suggest that E. pisi germlings release signal(s) at the time of maturation of their appressorium which is associated with the enhancement of inaccessibility. A further experiment demonstrated that the inaccessibility enhanced at the time of appressorial maturation persisted in the cells for at least 12 h. Based on these results, it was concluded that recognition of E. pisi by host cells occurred no later than the time of maturation of its appressorium, leading to temporary enhancement of inaccessibility.

Recognition of a pathogen and a nonpathogen by barley coleoptile cells (I). Cytoplasmic responses to the nonpathogen, Erysiphe pisi, prior to its penetration

Abstract Cytoplasmic responses of barley coleoptile cells to Erysiphe pisi , a nonpathogen for barley, were examined during the prepenetration stages of fungal infection using a time-lapse video system and oil droplets microinjected into cells. Conspicuous cytoplasmic strands were seen underneath and extending away from appressoria of the fungus. These strands became visible when a circular structure appeared in one of the appressorial lobes, a feature which was used to define appressorial maturity. Thereafter, active cytoplasmic streaming occurred near the appressoria. These cytoplasmic responses were evident 4–5 h prior to the actual penetration of the host cell by the fungus. To monitor the cytoplasmic activity of coleoptile cells, a droplet of silicon oil was injected into individual coleoptile cells before the fungus was inoculated, and the movement of the oil droplet was monitored with a video system. The droplet moved at 2–3 μm min −1 soon after injection but its velocity increased to 4–5 μm min −1 after a germling of the fungus was transferred onto the individual coleoptile cell. When the appressoria matured on the cells, the velocity increased dramatically to 73 μm min −1 . High velocities were then maintained, except for intermittent periods of slowing, until cytoplasmic aggregates were initiated beneath appressoria, when the velocity was reduced to 4–5 μm min −1 and the oil droplet remained near the appressorium. Although movement of the oil droplet did not correlate closely with rate of cytoplasmic streaming, the changes in droplet movement clearly indicated that cell cytoplasm responded to the presence of the fungus. These results suggest that appressoria of the fungus release a signal(s) upon maturation, 4–5 h prior to penetration, which induces physiological changes in the cytoplasm of the coleoptile cell.

Extracellular Materials of Fungal Structures: Their Significance at Prepenetration Stages of Infection

Historically, mechanisms of fungal infection have attracted plant pathologists, and our understanding of infection processes has been increased significantly for many disease interactions. Fungi generally undergo a series of distinct morphological changes before attempting host penetration. To date, most studies on infection processes deal with penetration and post-penetration stages of fungal development. However, several observations suggest that phenomena associated with initial stages of fungal contact on host surfaces play a critical role in host recognition, fungal differentiation, and success of infection [1, 13, 20, 26, 37, 38, 44, 64, 65, 73–76]. Since infection processes may be initiated soon after contact, prepenetration phenomena necessary for infection can easily be overlooked. One of these early prepenetration events is adhesion. The mechanisms of fungal adhesion are not as well understood as are those for bacterial adhesion [76]. Nicholson and Epstein [51] reviewed adhesion by plant pathogenic fungi and discussed mechanisms known to be involved in adhesion, as well as the broader topic of extracellular fungal matrices. The purpose of this review is to address cytological aspects of extracellular materials associated with fungal adhesion, and the subject of recognition between fungal and host cells.

Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XIII: Significance of haustorium formation by the pathogen Erysiphe graminis for induced accessibility to the non-pathogen E. pisi as assessed by nutritional manipulations

Erysiphe pisi , a non-pathogen of barley, never formed haustoria in fresh, healthy, barley coleoptile epidermal cells, even if cells were in constant contact with E. graminis germling structures, unless the attacked cell contained an haustorium of E. graminis . However, as reported in previous papers, where an haustorium of E. graminis was present in a cell, about 10–50% of E. pisi appressoria formed an haustorium in that cell depending on the timing of invasion by the latter fungus. To investigate the possible role of nutrient depletion in this induced accessibility to E. pisi , the nutritional status of host epidermal cells was manipulated in three ways: (a) tissues were inoculated with E. graminis at high infection densities so that almost all cells contained a haustorium; (b) all host cells adjacent to a cell containing a haustorium of E. graminis were killed by puncturing them with a microneedle; and (c) coleoptile tissues were starved for 6 days. In all three cases, development of secondary hyphae of E. graminis was fully or partially inhibited, unless exogenous glucose or sucrose was added, indicating that infected cells had insufficient sugar to support hyphal growth. In addition, treatments b and c induced accessibility to infection by E. pisi . In both cases, this accessibility was reversed by addition of 0·1 m glucose. These results indicate that accessibility of barley cells to E. pisi is induced by depletion of cell nutrients and that this occurs by prior infection of the cell and haustorium formation by E. graminis .

Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. VIII, Cytological evidence for suppressor(s) of host inaccessibility from Erysiphe graminis

Abstract The possibility that a suppressor(s) of inaccessibility is released by Erysiphe graminis was examined using a system that involved the coordinate inoculation of a single barley coleoptile cell with E. graminis and E. pisi (a nonpathogen). Partially dissected coleoptiles were inoculated with E. pisi conidia. Germinating conidia of this fungus were removed from the host surface with a micromanipulator at or after maturation of the appressorium. Germinating conidia of E. graminis which had been inoculated onto another coleoptile were then transferred to the coleoptiles inoculated initially with E. pisi . This was done so that the E. graminis germling would attempt to penetrate the same cell from which E. pisi germling had been removed. The E. graminis transfer was made before, at, or after the time of maturation of the E. pisi appressorium. Results showed that the induced inaccessibility of cells that suppress the penetration by E. graminis was expressed only when E. graminis was transferred to the cells at or after the maturation of the E. pisi appressorium. If E. graminis was transferred before the time of the maturation of an E. pisi appressorium it suppressed the enhancement of inaccessibility caused by E. pisi . In contrast, if the E. pisi appressorium was allowed to mature prior to the transfer of E. graminis , inaccessibility could not be suppressed. A further transfer experiment revealed that the continued presence of an E. graminis germling on a host cell suppressed the inaccessibility caused by E. pisi on the same cell. The results support the hypothesis that E. graminis releases a suppressor(s) which blocks the effect of the E. pisi elicitor.

Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta) and transient gene expression using the rbcS gene promoter

We isolated two different genomic DNAs (UprbcS1 and UprbcS2) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and portions of the 5′- and 3′-flanking regions from sterile Ulva pertusa Kjellman. The UprbcS1 and UprbcS2 genes had three introns in the coding region. Each predicted UprbcS polypeptide was a 180-amino-acid (AA) residue including a 38-AA transit peptide, although the 104th AA residue was replaced. The nucleotide sequences of UprbcS cDNAs isolated from a cDNA library corresponded to that of the UprbcS1 gene, suggesting that the UprbcS1 gene was predominantly expressed in sterile U. pertusa compared to UprbcS2. Southern blot analysis showed that each UprbcS gene was a single-copy gene in the sterile U. pertusa genome. Northern hybridization indicated that the expression of UprbcS was induced and repressed by dark and light treatments, respectively. When sterile U. pertusa cells were transformed with an expression vector containing the UprbcS1 promoter and terminator sequences fused with the green fluorescent protein (GFP) gene, GFP fluorescence was observed in the cells transformed. These results suggest that the UprbcS1 gene promoter is light regulated and highly active in the sterile U. pertusa cells and is available for genetic transformation system in the alga.