Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.     

Changes in soluble proteins in potato leaves infected with Phytophthora infestans

A new protein was observed in the electrophoretic gel band ofleaves of cultivar susceptible to Phytophthora infestans, whenthe DNA fraction of a resistant hybrid was applied to the leaves.The Rf value of this band coincided with that of the hybrid6 hr after die inoculation of a race of P. infestans to whichit was resistant. ¹Present adress: Laboratory of Plant Pathology, College of Agriculture.Kyoto University, Kyoto, 606) Japan. ²His former family name was Nakao. (Received December 19, 1975; )     

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.     

Pathogen-induced plant proteins

Pathogen-induced plant proteins are classified by their functional characteristics: (a) involvement in plant cell signaling; (b) inhibition of enzymes excreted by the pathogens; (c) stabilization of plant cell walls or ability to trigger apoptosis; (d) enzymatic activity producing lysis of cell walls of pathogenic fungi and bacteria; (e) enzymatic activity in metabolic pathways of phenylpropane and terpene phytoalexins; and (f) ability to affect the pathogens directly, by disturbing the function of their cell membranes or by inactivating their ribosomes. Examples of transgenic plants with increased immunity against pathogens are also provided.     

Heteroplasmosis in Phytophthora infestans

APCR and monozoospore plating was used to demonstrate a simultaneous presence of the mitochondria with mitochondrial DNA of haplotypes ILa and IIa in the mycelium of several Phytophthora infestans strains.     

Cyst germination proteins of the potato pathogen Phytophthora infestans share homology with human mucins

We have cloned genes of Phytophthora infestans, the causal agent of potato late blight, that are activated shortly before the onset of invasion of the host tissue. The three genes isolated appear to be arranged in a genomic cluster and belong to a small polymorphic gene family. A conspicuous feature of the deduced proteins is an internal octapeptide repeat with the consensus sequence TTYAP TEE. Because of this structural motif, these novel P. infestans proteins were named Car (Cyst-germination-specific acidic repeat) proteins. One of the genes, car90, codes for 1,489 amino acids including 120 octapeptide tandem repeats. Car proteins are transiently expressed during germination of cysts and formation of appressoria and are localized at the surface of germlings. The structural motif of tandemly repeated oligopeptides also occurs in a prominent class of proteins, the mucins, from mammals. The P. infestans Car proteins share 51% sequence homology with the tandem repeat region of a subfamily of human mucins. According to the physiological functions ascribed to mucins, we suggest that Car proteins may serve as a mucous cover protecting the germling from desiccation, physical damage, and adverse effects of the plant defense response and may assist in adhesion to the leaf surface.     

Genetics of metalaxyl resistance in Phytophthora infestans.

Several sexual crosses involving isolates of Phytophthora infestans of diverse sensitivities to metalaxyl were studied. Metalaxyl sensitivity was determined by comparing the growth of an isolate on metalaxyl-amended agar medium (5 microg/ml) with growth on medium containing no metalaxyl. When both parents had the same phenotype for metalaxyl sensitivity (both resistant or both sensitive), all F1 progeny had the parental phenotype. In two crosses (75 and 76) each involving one sensitive and one resistant parent, however, the progeny segregated 1:1, suggesting that the common resistant parent (Bg8) was heterozygous for metalaxyl sensitivity. When an F2 progeny was constructed from resistant F1 isolates in cross 76, the progeny segregated 1:3 (sensitive:resistant), indicating that metalaxyl resistance in Bg8 is conferred by a single dominant gene. Variation in the progeny sensitivity appears to involve minor genes. A correlation study between metalaxyl resistance and fitness components did not reveal any association.     

Antifungal activity of oosporein from an antagonistic fungus against Phytophthora infestans

An antifungal metabolite, oosporein, was isolated from the culture of Verticillium psalliotae that produced the antagonistic effects on Phytophthora infestans. Oosporein exhibited a significant growth-inhibitory effect on P. infestans in comparison with other phytopathogenic fungi.     

Phenotypic markers of Phytophthora infestans

Abstract

Phytophthora infestans is one of the most destructive pathogens of potato and causal agents of notorious disease late blight. Different chemicals are used to control the pathogen of late blight but the most commonly used is metalaxyl; its extensive use of has caused decreased sensitivity in the P. infestans population. The metalaxyl sensitivity of the Pakistani population of P. infestans is investigated in the present study. For this purpose, 178 isolates of P. infestans were obtained from the lesions of diseased potato leaves and stems, and samples were collected from the different potato-growing areas of Pakistan, where late blight is a problem. Sensitivity of the isolates of P. infestans was investigated by metalaxyl sensitivity test and with the help of test isolates were divided into three categories, i.e. sensitive, intermediate and resistant, based on their Co-efficient of mycelial growth inhibition (CMGI) values. During the study, highest percentage (50.17%) of resistant isolates was observed in the population of Punjab (zone 2), whereas the lower percentage (33.33%) was observed in the population of Swat valley (zone 6b). In the present study, it was discovered that P. infestans late blight-causing fungus has adopted more resistance against metalaxyl because of its wide use.     

Exoproteinases of the Oomycete Phytophthora infestans

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown (by SDS-PAGE in the presence of gelatin) to include at least six components differing in molecular weight. Inhibitory analysis and studies of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases (specific for trypsin and subtilisin) and metalloproteinases. Their activity is suppressed by proteinase-inhibiting proteins from potato tubers. It is suggested that exoproteinases of P. infestans may be the metabolic target for natural proteinase inhibitors from potato.     

Exoproteinases of the oomycete Phytophthora infestans

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown by SDS-PAGE with the presence of gelatin to include at least six components differing in molecular weight. Inhibitory analysis and study of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases specific to trypsin and subtilisin and metalloproteinases. Their activity is suppressed by proteinase-inhibitor proteins from potato tubers. It is suggested that P. infestans exoproteinases may be the metabolic target for natural proteinase inhibitors from potato.