Richard J. Howard

Adhesion Pad Formation and the Involvement of Cutinase and Esterases in the Attachment of Uredospores to the Host Cuticle.

We have investigated the basis of adhesion of uredospores of the obligately parasitic rust fungus Uromyces viciae-fabae to leaves of its broad bean host. Upon contact with an aqueous environment, spores form a structure that we have termed an adhesion pad. The adhesion pad is formed by both living and autoclaved spores, but only adhesion pads formed by living spores adhered to the cuticle of leaves of the host plant. Treatment of living spores with the serine-esterase inhibitor diisopropyl fluorophosphate prevented the adhesion of the pad to the leaf surface, suggesting a functional role for esterase or cutinase in the process of adhesion. A cutinase and two nonspecific serine-esterases were found to be localized on the surface of spores. These enzymes were released rapidly from the spore surface upon contact with an aqueous environment. The addition of the cutinase and the nonspecific esterases to autoclaved spores restored their ability to adhere to the host cuticle. Thus, whereas pad formation appears to be a passive response to the aqueous environment, the actual adhesion of pads to the host cuticle appears to depend on the cutinase and esterases associated with the spore surface. These results suggest a new role for cutinases and serine-esterases in the fungal infection process.

Cell Number Regulator1 Affects Plant and Organ Size in Maize: Implications for Crop Yield Enhancement and Heterosis

This study describes the isolation and characterization of the maize CNR gene family. It provides evidence that CNR1 affects plant size as a negative regulator of cell number, suggesting potential for application to crop improvement. Genes involved in cell number regulation may affect plant growth and organ size and, ultimately, crop yield. The tomato (genus Solanum) fruit weight gene fw2.2, for instance, governs a quantitative trait locus that accounts for 30% of fruit size variation, with increased fruit size chiefly due to increased carpel ovary cell number. To expand investigation of how related genes may impact other crop plant or organ sizes, we identified the maize (Zea mays) gene family of putative fw2.2 orthologs, naming them Cell Number Regulator (CNR) genes. This family represents an ancient eukaryotic family of Cys-rich proteins containing the PLAC8 or DUF614 conserved motif. We focused on native expression and transgene analysis of the two maize members closest to Le-fw2.2, namely, CNR1 and CNR2. We show that CNR1 reduced overall plant size when ectopically overexpressed and that plant and organ size increased when its expression was cosuppressed or silenced. Leaf epidermal cell counts showed that the increased or decreased transgenic plant and organ size was due to changes in cell number, not cell size. CNR2 expression was found to be negatively correlated with tissue growth activity and hybrid seedling vigor. The effects of CNR1 on plant size and cell number are reminiscent of heterosis, which also increases plant size primarily through increased cell number. Regardless of whether CNRs and other cell number–influencing genes directly contribute to, or merely mimic, heterosis, they may aid generation of more vigorous and productive crop plants.

Reef coral fluorescent proteins for visualizing fungal pathogens.

The fluorescent proteins AmCyan, ZsGreen, ZsYellow, and AsRed, modified versions of proteins identified recently from several Anthozoa species of reef corals, were expressed for the first time in a heterologous system and used for imaging two different fungal plant pathogens. When driven by strong constitutive fungal promotors, expression of these reef coral fluorescent proteins yielded bright cytoplasmic fluorescence in Fusarium verticillioides and Magnaporthe grisea, and had no detectable effect on either growth rate or the ability to cause disease. Differential intracellular localization of the fluorescent proteins resulted in the discrimination of many subcellular organelles by confocal and multi-photon microscopy, and facilitated monitoring of such details as organelle dynamics and changes in the permeability of the nuclear envelope. AmCyan and ZsGreen were sufficiently excited at 855 and 880 nm, respectively, to allow for time-resolved in planta imaging by two-photon microscopy.

Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing

Abstract. High-pressure freezing and freeze substitution were used to prepare leaves of rice (Oryza sativa L.) for ultrastructural analysis. Under these preparative conditions, plastid-derived stroma-containing protuberances were preserved and described with the electron microscope for the first time. Similar protuberances were observed previously only in living cells examined with the light microscope. Infoldings of the chloroplast inner envelope were a prominent ultrastructural feature of protuberances. Infoldings were also observed in the main body of the chloroplasts and sometimes appeared contiguous with thylakoid membranes. Protuberances also contained infoldings in the form of bifurcated tubules. Apparent interconnection between protuberances of adjacent plastids was observed only in one instance. A distinct gradient in the staining density of thylakoid lumina appeared to be a function of grana position and orientation relative to the cell wall. Immunocytochemistry was used to determine that the stroma within protuberances contained 1,5-bisphosphate carboxylase/oxygenase enzyme.

Biology of Maize Kernel Infection by Fusarium verticillioides

Fusarium kernel rot disease starburst symptomatology was characterized fully for the first time. Two maize lines were hand pollinated and inoculated, using a fluorescent protein-expressing transformant of the fungal pathogen Fusarium verticillioides, by introduction of a conidial suspension through the silk channel of intact ears. Microscopy was used to identify the infection court and document initial stages of kernel colonization and subsequent manifestation of macroscopic symptoms. The fungus entered kernels of susceptible line AD38 via an open stylar canal and spread extracellularly and over the kernel through the nucellus region, sporadically entering pericarp and filling the long thick-walled mesocarp cells. Hyphae spread within pericarp from cell to cell via pits, colonizing files of host cells by growing both up and down the kernel in a radial pattern that preceded macroscopic symptom development. The starburst symptom developed subsequently, and mirrored colonization exactly, when there was extensive dissolution of the thick walls of pericarp cells. Line HT1 exhibited a closed stylar canal phenotype and was not susceptible-except when the pericarp surface was breached mechanically. We hypothesize the passive movement of conidia along the surface of silks, perhaps via capillarity, as a possible mechanism for pathogen access to the infection court.

Gland Development on Leaf Surfaces of Nepeta racemosa

Trichomes present on the leaf surface of catmint (Nepeta racemosa L.) were examined using both scanning and transmission electron microscopy. Three types of trichomes were observed: (a) multicellular, uniseriate, nonglandular hairs, (b) small capitate glands with two cells in the head, and (c) large, peltate glands with four secretory/head cells. Secretory cells of capitate glands exhibited typical transfer cell morphology, with an abundance of RER and Golgi bodies, indicative of secretion of mainly hydrophilic substances. Mature peltate glands exhibited a distended cuticle and large subcuticular cavity, presumably containing the essential oils. Morphologically well-developed peltate glands were observed on young leaf primordia, with development occurring earlier at the leaf apex and along the midvein than at the leaf margins or base. Four major stages in the development of the peltate gland secretory cells were identified. The first stage was marked by highly vacuolated cytoplasm and normal distribution of subcellular organelles. The second stage, during which there was substantial accumulation of material in the subcuticular cavity, was marked by an abundance of tubular endomembrane elements in proximity to plastids with little internal differentiation. Retraction from the cell wall and an unusual scalloped appearance to the plasmalemma characterized a third stage. Presence of a large subcuticular cavity, an exaggerated retraction of the plasmalemma, and a band of peripheral mitochondria were typical of a fourth, presumably mature stage.

Satellite Spitzenkörper in growing hyphal tips

SummaryGrowing hyphal tips of higher fungi contain an organized assemblage of secretory vesicles and other cell components collectively known as the Spitzenkörper. Until now, the Spitzenkörper has been portrayed as a single spheroid complex located near the apical cell wall. This study demonstrates the occurrence of multiple Spitzenkörper in growing hyphal apices imaged by video-enhanced phase-contrast microscopy. In addition to the main Spitzenkörper, smaller satellite Spitzenkörper arise a few micrometers behind the apical pole. Four developmental stages were identified: (a) the satellites first appeared as faint phase-dark plaques next to the plasma membrane, (b) gradually increased in size and assumed an ovoid profile, (c) they migrated to the hyphal apex, and (d) finally they merged with the main Spitzenkörper. After the merger, the main Spitzenkörper temporarily increased in size. Satellites were observed in 14 fungi, most of which had relatively large (5–10 μm diam.), fast-growing hyphae (2–33 μm/min elongation rate). The average frequency of in-focus satellites was 7+/min forFusarium culmorum and 11+/min forTrichoderma viride. As with the main Spitzenkörper, satellites were present only in growing cells. They were transient and remained visible for 3–8 s before merging with the main Spitzenkörper. Within the hyphae, satellites travelled up to six times faster than the average cell elongation rate. Multiple satellites sometimes occurred simultaneously; up to three were seen within a hyphal apex at the same time. Localized cell enlargement occurred next to stationary satellites, suggesting that satellite Spitzenkörper are functional as sources of new cell surface before they reach the main Spitzenkörper; therefore, they account for some variations in the profiles of the growing hyphae. By electron microscopy, satellites consisted of small clusters of apical vesicles surrounding a group of microvesicles located next to the plasma membrane. The identification and behavior of the satellites represent clear evidence of directional mass transport of vesicles toward the hyphal apex. Our observations indicate that satellites are a common phenomenon in growing hyphal apices of septate fungi and that they contribute to growth of the hyphal apex.

Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents

Summary.Microtubule dynamics were examined in live cells of the fungal plant pathogen Magnaporthe grisea transformed for constitutive expression of a fusion protein containing enhanced yellow-fluorescent protein and a Neurospora crassa benomyl-resistant allele of β-tubulin. Transformants retained their ability to differentiate appressoria and cause disease but remained sensitive to benomyl. Linear microtubule arrays and low-level cytoplasmic fluorescence were observed in vegetative hyphae, conidia, germ tubes, and developing appressoria. Fluorescence within nuclei was conspicuously absent during interphase but increased rapidly at the onset of mitosis. Treatment with either benomyl or griseofulvin resulted in the appearance of prominent brightly fluorescent aggregates, including a large aggregate near the apex, with the concomitant disappearance of most cytoplasmic microtubules. Electron microscope imaging of treated cells indicated that the aggregates lacked any obvious profiles of intact microtubules. During these treatments, hyphal tip cells continued to elongate in a nonlinear and aerial fashion at a much slower rate than untreated cells. With subsequent removal of griseofulvin, distal aggregates disappeared rapidly but the apical aggregates persisted longer. Treatment with latrunculin A caused hyphal tip swelling without apparent effect on linear microtubule arrays. Simultaneous treatment with griseofulvin and latrunculin A resulted in depolymerization of microtubules and a cessation of growth, but near-apical fluorescent aggregates were not observed.

Utility of cytoplasmic fluorescent proteins for live-cell imaging of Magnaporthe grisea in planta.

The subcellular expression patterns and fluorescence intensities of cytoplasm-targeted, constitutively expressed blue-, cyano-, green-, yellow- and red-fluorescent protein were assessed in a number of transformants of the blast pathogen, Magnaporthe grisea. All transformants grew normally, remained pathogenic on barley, and, except for those expressing blue fluorescent protein, exhibited significant cytoplasmic fluorescence. The exceptionally intense brightness of some strains proved very useful for laser scanning confocal microscope imaging during invasion of host tissues. Acquisition of three-dimensional data sets from intact, individual, pathogen encounter sites in planta were generated during the time course of pathogenesis using non-invasive optical sectioning methods. Confocal and multiphoton microscopy imaging in conjunction with fluorescent protein expression allowed for the real time documentation of fungal colonization within plant cells and tissues with remarkable ease. These methods constitute valuable new tools for the investigation of plant disease.

Cytology of fungal pathogens and plant-host interactions.

Imaging plays a unique role in fungal cell biology and phytopathology by allowing for the documentation of molecular structure in individual fixed and living cells. Advances in fluorescence laser techniques, including confocal and multiphoton microscopy, are opening new avenues for cellular exploration. These techniques hold tremendous potential for studies of host-pathogen interactions including the use of genetically encoded markers such as green fluorescent protein, in situ hybridization and fluorescence resonance energy transfer.