Dm3 is one member of a large constitutively expressed family of nucleotide binding site-leucine-rich repeat encoding genes

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.     

Nuclear localization of protein phosphatase 5 is dependent on the carboxy-terminal region

Endogenous and overexpressed protein phosphatase 5 (PP5) localizes to the nucleus and cytoplasm of HeLa cells, while the overexpressed TPR domain of PP5 is restricted to the cytoplasm. Deletion and mutational analysis of human PP5 demonstrates that the C-terminal amino acids 420-499 are essential for nuclear localization and PP5 activity is not required. Since the phosphatase domain terminates at 473, these studies suggest that the highly conserved section (476-491) with the eukaryotic consensus FXAVPHPXPhiXPMAYAN is required for nuclear localization of PP5. Bacterially expressed PP5 is inhibited by several tumor promoters but not by the anticancer drug fostriecin.     

Protein phosphatase-1 binding to scd5p is important for regulation of actin organization and endocytosis in yeast

SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast. Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes. PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the -1 or -2 position). Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276). Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs. Therefore, the consequence of mutating these two potential PP1-binding sites was examined. Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth. Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization. Overexpression of GLC7 suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype. These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.     

Ectopic expression of inhibitors of protein phosphatase type 1 (PP1) can be used to analyze roles of PP1 in Drosophila development

We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGF beta superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.     

The chaperone-like properties of mammalian inhibitor-2 are conserved in a Drosophila homologue

Phosphatase inhibitor-2 (I-2) is a mammalian phosphoprotein that binds to the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1c) and inhibits its activity in vitro. Recombinant PP1c differs from native PP1c in several biochemical criteria, including the requirement for Mn(2+), sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity. I-2 can convert recombinant PP1c into a native-like activity in vitro. It has therefore been suggested that I-2 may act as a molecular chaperone for PP1 in vivo. We have identified a Drosophila homologue (I-2Dm) in a two-hybrid screen for PP1c-binding proteins. The sequence of I-2Dm is 35% identical with that of I-2, whereas the catalytic subunits themselves are >85% identical in flies and humans; however, we show that many biochemical properties of I-2 are conserved. Like I-2, I-2Dm can convert recombinant PP1c to a native-like activity. This strongly suggests that this ability is an essential, conserved role of I-2 and I-2Dm.     

Phosphorylation and functions of inhibitor-2 family of proteins

Protein phosphatase-1 (PP1) is an essential protein Ser/Thr phosphatase that is extraordinarily conserved from yeast to human, and Inhibitor-2 (I-2) is the most ancient of the heat-stable proteins specific for PP1. We identified novel I-2 homologues in Caenorhabditis elegans (Ce) and Xenopus laevis (Xe) and compared them to the I-2 proteins from Homo sapiens (Hs), Saccharomyces cerevisiae (GLC8), and Drosophila melanogaster (Dm). The Ce I-2 and Dm I-2 showed the highest potency inhibition of rabbit PP1 with IC50 near 5 nM compared to Hs I-2 and Xe I-2 with IC50 between 10 and 50 nM and GLC8 with >100-fold lower activity. Inhibition of PP1 bound to Nek2 kinase activated the kinase to phosphorylate a C-Nap1 domain substrate. All the species of I-2 except GLC8 activated the Nek2::PP1 to the same extent as microcystin-LR. Only Hs I-2 and Xe I-2, not the I-2 proteins more divergent in sequence, directly activated human Aurora-A kinase. Various species of I-2 have a common PxTP phosphorylation site that showed a wide range of reactivity with GSK3, ERK, or CDC2/cyclinB1 kinases. The Suc1 subunit of CDC2/cyclinB1 enhanced reactivity with I-2, consistent with this being a site of mitotic phosphorylation. The results show species specificity among the I-2 family within the context of conserved PP1 inhibitory activity and variable phosphorylation by Pro-directed kinases.     

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

The disease resistance gene Dm3 is infrequent in natural populations of Lactuca serriola due to deletions and frequent gene conversions at the RGC2 locus

Resistance genes can exhibit heterogeneous patterns of variation. However, there are few data on their frequency and variation in natural populations. We analysed the frequency and variation of the resistance gene Dm3, which confers resistance to Bremia lactucae (downy mildew) in 1033 accessions of Lactuca serriola (prickly lettuce) from 49 natural populations. Inoculations with an isolate of Bremia lactucae carrying avirulence gene Avr3 indicated that the frequency of Dm3 in natural populations of L. serriola was very low. Molecular analysis demonstrated that Dm3 was present in only one of the 1033 wild accessions analysed. The sequence of the 5' region of Dm3 was either highly conserved among accessions, or absent. In contrast, frequent chimeras were detected in the 3' leucine-rich repeat-encoding region. Therefore low frequency of the Dm3 specificity in natural populations was due to either the recent evolution of Dm3 specificity, or deletions of the whole gene as well as variation in 3' region caused by frequent gene conversions. This is the most extensive analysis of the prevalence of a known disease resistance gene to date, and indicates that the total number of resistance genes in a species may be very high. This has implications for the scales of germplasm conservation and exploitation of sources of resistance.     

Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa)

Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion.     

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.     

Actin gene expression is modulated by ecdysterone in a Drosophila cell line

The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.