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The y-1 mutant of Chlamydomonas reinhardtii is defective in the conversion of protochlorophyllide (Pchlide) to chlorophyllide in the dark. Aerobic delta-aminolevulinic acid (ALA) feeding of y-1 cells causes protoporphyrin monomethyl ester (PME) to accumulate in addition to increased levels of Pchlide. y-1 cell homogenates are not capable of methylating protoporphyrin (PROTO) to form PME but can methylate magnesium protoporphyrin (MgP) to form magnesium protoporphyrin monomethyl ester (MgPME). Anaerobic ALA feeding of y-1 causes concomitant accumulation of PME and MgPME. y-1 cells treated with alpha,alpha'-dipyridyl (DP) accumulate MgPME but not PROTO or PME. A mutant strain (bme) of Chlamydomonas has been isolated which has very little chlorophyll and accumulates PME. bme Cell homogenates can methylate MgP but not PROTO. We propose that: (a) in Chlamydomonas, PME is the initial breakdown product of MgPME; (b) both the breakdown of MgPME to PME and the conversion of MgPME to Pchlide require O(2); (c) the breakdown of MgPME to PME appears to require Fe; and (d) the PME accumulated in the bme mutant is the result of an increased breakdown of MgPME.

A group of chlorophyll deficient mutants (br s mutants) of Chlamydomonas accumulates protoporphyrin and has poorly developed chloroplast membrane systems (Wang et al. 1974). In order to determine whether a poorly developed chloroplast membrane system is the reason for, or the result of, the inability of the br s mutants to metabolize protoporphyrin to chlorophyll, a second mutation was selected which restored chlorophyll synthesis in br s mutants. One such double mutant (br s-2 g-4) was analyzed. The double mutant br s-2 g-4 has partially restored chlorophyll synthesis, but has defective photosystem II and photosystem I electron transport as well as abnormal chloroplast ultrastructure. Since these defects are not present in cells carrying only the g-4 mutation, they are presumed to be caused by the br s-2 mutation. It is concluded that a defect in chloroplast membrane development resulting from the br s-2 mutation causes an apparent defect in magnesium chelation by protoprophyrin. This is consistant with evidence that chlorophyll biosynthesis from magnesium protoporphyrin to chlorophyll takes place on the chloroplast membranes.

The heterokaryotic and vegetative diploid phases of Magnaporthe grisea, a fungal pathogen of grasses, have been characterized. Prototrophic heterokaryons form when complementary auxotrophs are paired on minimal medium. Hyphal tip cells and conidia (vegetative spores) taken from these heterokaryons are auxotrophs with phenotypes identical to one or the other of the parents. M. grisea heterokaryons thus resemble those of other fungi that have completely septate hyphae with a single nucleus per cell. Heterokaryons have been utilized for complementation and dominance testing of mutations that affect nutritional characteristics of the fungus. Heterokaryons growing on minimal medium spontaneously give rise to fast-growing sectors that have the genetic properties expected of unstable heterozygous diploids. In fast-growing sectors, most hyphal tip cells are unstable prototrophs. The conidia collected from fast-growing sectors include stable and unstable prototrophs, as well as auxotrophs that exhibit a wide range of phenotypes, including many recombinant classes. Genetic linkage in meiosis has been detected between two auxotrophic mutations that recombine in vegetatively growing unstable diploids. The appearance of recombinants suggests that homologous recombination occurs during vegetative growth of M. grisea. No interstrain barriers to heterokaryosis and diploid formation have been detected. The mating type of the strains that are paired does not influence the formation of heterokaryons or diploids.

The aerial parts of plants are covered by the cuticle which forms the boundary layer at which microbes come into contact with the plant. The cuticle is composed of an insoluble structural polymer, cutin, which is embedded in a complex mixture of soluble lipids collectively called wax (1,2). Cutin is composed of interesterified hydroxy and hydroxyepoxy fatty acids primarily derived from palmitic, oleic, and linoleic acids (Fig. 1). The component fatty acids are held together by ester bonds between the primary as well as secondary hydroxy groups and the carboxyls. The cutin polymer constitutes the major physical barrier to penetration of fungi into the aerial parts of plants (3). The cutin barrier is attached to a pectinaceous polymeric layer which may also serve as a barrier to penetration by pathogens. In this paper, we will briefly review progress recently made in our understanding of the role of fungal cutinases and pectinases in the interaction between pathogenic fungi and their host plants. We also describe a recent discovery that a phyllospheric bacterium which cohabits with an apparently nitrogen-fixing bacterium generates an extra-cellular cutinase and thus can provide a carbon source while receiving fixed N2 from the cohabiting partner.

Since indirect experimental evidence suggested that penetration of Fusarium solani f. sp. pisi into its host (Pisum sativum) involved pectin-degrading enzymes (W. Koller, C. R. Allan, and P. E. Kolattukudy (1982) Physiol. Plant Pathol.20, 47–60) , direct tests were made for the production of such degradative enzymes by this pathogen. When the organism was grown on pectin, a pectate lyase (EC 4.2.2.2) was released into the media. This lyase was purified to apparent homogeneity from the culture filtrate by a two-step process involving passage through DEAE-Sephacel followed by hydrophobic interaction chromatography on octyl-Sepharose. The enzyme cleaved polygalacturonate chains in an endo fashion. The molecular mass of the mature extracellular form of this enzyme was estimated to be 26 kDa. The isoelectric point of the enzyme was 8.3 and the optimum pH for activity was 9.4. Calcium was required for activity and evidence is presented that calcium probably interacts with the substrate rather than the enzyme. When antibodies prepared against this enzyme were used for Western blot analysis of the extracellular culture fluid, a single band was observed at 26 kDa. Following in vitro translation of poly(A)+ RNA, a 29-kDa precursor polypeptide was precipitated by the antibodies. Antibodies inhibited both the catalytic activity of the enzyme and the ability of the fungus to infect pea stems, strongly suggesting that this lyase is involved in pathogenesis.