4Prasher (42) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria and Caenorhabditis elegans cells in culture (10, 31), followed by yeast (24,39), mammals (40), Drosophila (66), Dictyostelium (23,30), plants (28,49), and filamentous fungi (54). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (␤-glucuronidase, ␤-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation of gfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17 and 67), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 (67).Limitations on GFP as a reporter for some applications are its low turnover rate, 2-h lag time for autoactivation of its chromophore, improper folding at high temperatures (37°C), which results in nonfluorescent and insoluble forms of the protein, and requirement for oxygen, which is not present in equal concentrations in all subcellular locations or cell types (reviewed in references 17 and 67). These characteristics of GFP, however, have not posed a problem for many applications, and mutant forms of GFP that have an ability to fold properly at high temperatures, increased solubility and fluorescence, reduced photobleaching (16, 17, 51), and reduced half-lives (1) have been developed. Coupled with fluorescenceactivated cell sorting, confocal microscopy or quantitative image analysis techniques, GFP technology can be used to isolate transformed cells or specific cell types from populations of cells (14), to quantify gene expression of individual cells within whole organisms (8), or to assess the dispersal and biomass of organisms in complex environments, such as in animal or plan...

Summary Host‐selective toxins (HSTs) are effectors produced by some necrotrophic pathogenic fungi that typically confer the ability to cause disease. Often, diseases caused by pathogens that produce HSTs follow an inverse gene‐for‐gene model where toxin production is required for the ability to cause disease and a single locus in the host is responsible for toxin sensitivity and disease susceptibility. Pyrenophora tritici‐repentis represents an ideal pathogen for studying the biological significance of such inverse gene‐for‐gene interactions, because it displays a complex race structure based on its production of multiple HSTs. Ptr ToxA and Ptr ToxB are two proteinaceous HSTs produced by P. tritici‐repentis that are structurally unrelated and appear to evoke different host responses, yet each toxin confers the ability to cause disease. This review will summarize the current knowledge of how these two dissimilar HSTs display distinct modes of action, yet each confers pathogenicity to P. tritici‐repentis.

ToxB, a gene that encodes a 6.6-kDa host-selective toxin (HST), is present in several races of the wheat pathogen Pyrenophora tritici-repentis. To learn more about the multiple ToxB open reading frames (ORFs), six of the estimated nine copies from a race 5 isolate were cloned and analyzed. All six copies of ToxB have identical 261-bp ORFs and thus encode the same form of Ptr ToxB. Sequence analysis of regions flanking the cloned ToxB loci revealed that the majority of loci are associated with portions of retrotransposons and a transposon-like sequence. Data indicate that ToxB loci reside on two chromosomes, 3.5 and 2.7 Mb, with the majority of copies located on the 2.7 Mb chromosome. A related gene, referred to as toxb, from a nonpathogenic race 4 isolate was also cloned and characterized. This is interesting because, until now, HST genes have only been found in toxin-producing, pathogenic isolates of plant pathogenic fungi. The toxb gene from nonpathogenic isolates is 86% similar to ToxB, and data suggest that toxb is a single-copy gene. No toxb transcript was detected under culture conditions that favor the expression of ToxB; therefore, these genes differ in their transcriptional regulation.

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