Pumilio homologue from saprolegnia parasitica specifically expressed in undifferentiated spore cysts

Abstract

The expression of spore-specific marker transcripts at different stages of the asexual life cycle of Saprolegnia parasitica was analyzed. One of the markers, designated puf1, was found to be expressed transiently upon each of several cycles of zoospore encystment and reemergence. The transcript is induced immediately upon zoospore encystment and is rapidly lost when a cyst is triggered to germinate. In nongerminating cysts, puf1 is maintained until a time point when the cysts can no longer be triggered to germinate and thus have become determined for zoospore reemergence. The results show that the cyst stage has two phases, of about equal duration, which are physiologically and transcriptionally distinct and that the transcriptional machinery of oomycetes is also active in nongerminating spores. puf1 encodes a putative mRNA binding protein belonging to a conserved class of proteins including the Drosophila melanogaster Pumilio protein, Caenorhabditis elegans FBF, and Saccharomyces cerevisiae Puf5, all of which are involved in regulation of gene expression by post-transcriptional mechanisms.

FIG. 1.

Confirmation of stage-specific transcripts by Northern blotting of total RNA. (a) Expression of puf1 in spores, cysts induced to germinate (20 min after induction), germlings (90 min after induction), nongerminating cysts collected 60 min after encystment, young (4-h) mycelium from germinating cysts, and 3-day-old mycelium. The blot was hybridized simultaneously with probes puf1f-sma (upper blot) and tef1 (lower blot). Ribosomal bands were stained with ethidium bromide. (b) Expression of sst2, sst15, and mst3 in zoospores, cysts induced to germinate (15 min after induction), germlings (90 min after induction), and 3-day-old mycelium. The blots were reprobed with tef1.

FIG. 2.

A selection of members of the Puf family. Characteristic of this family is an RNA binding domain consisting of eight repeats and two flanking sequences, Csp1 and Csp2. The eight repeats are well conserved within the Puf family, while the Csp motifs vary between different subclasses. The N termini of the proteins consist of a nonconserved domain of unknown function. Percent identities and similarities between the RNA binding domains of the different proteins and that of Puf1 are indicated.

FIG. 3.

Expression of puf1 in sporulating mycelium harvested 0, 2, 4, 6, and 8 h after washing. (a) Interference contrast microscopy of mature zoosporangia at 8 h after induction of sporulation, with visible spores ready to be released. (b) Northern blot of total RNA hybridized with probe puf1f-sma (upper panel). The next three panels, from top to bottom, show ribosomal bands stained with ethidium bromide and Northern blots hybridized with sst2 and tef1, respectively.

FIG. 4.

puf1 is induced transiently upon encystment. (a) RT-PCR with gene-specific primers for puf1 (upper panels), sst2 (middle panels), and sst15 (lower panels) on total RNA from the first zoospore generation; from cysts harvested 0, 15, 30, 60, 90, and 120 min after vortexing; and from the second zoospore generation. RT-PCR was also performed with the same primers on total RNAs from the first, second, third, and fourth cyst generations. (b) Percentages of cysts that have released a second zoospore generation at different time points after encystment.

FIG. 5.

Disappearance of spore-specific transcripts upon germination. (a) Length of germ tubes in cysts 0 to 40 min after induction of germination by vortexing together with a nutrient trigger. (b) RT-PCR with primers for puf1, sst2, sst15, and actin on total RNA from cysts harvested 5 to 40 min after induction of germination.

FIG. 6.

(a) Percentage of cysts germinating in response to a nutrient trigger added at different time points after encystment by vortexing. (b) Germ tube length in cysts triggered to germinate by addition of a nutrient trigger (PG1) at different time points after encystment by vortexing: ▪, PG1 added at 0 min; ▴, PG1 added at 25 min; •, PG1 added at 50 min; ⧫, PG1 added at 75 min. (c) Disappearance of puf1 upon delayed induction of germination. RT-PCR with universal Puf primers was done with nongerminating cysts collected at 15, 30, or 55 min after encystment by vortexing or with cysts triggered to germinate by addition of PG1 15 min after vortexing followed by an additional 15 or 40 min of incubation.

FIG. 7.

Induction of puf1 results from de novo synthesis. RT-PCR was done on cDNA primed with primer T₃₀MC (left) or the universal Puf primer (right). PCR with universal Puf primers (upper panel) and sst15-specific (spec) primers (lower left panel) was done with zoospores, cysts 15 min after vortexing, cysts 100 min after vortexing, or germlings 1 h after addition of a nutrient trigger.