Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and beta-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.

Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and β-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.

We transformed SR1 tobacco with a cDNA fragment of the potato virus Y strain N (PVYN) 605 genome which contained the 3' end of the NIb polymerase gene, the coat protein (CP) coding sequence, and most of the viral 3' untranslated region. Complete resistance to several PVY strains was obtained in plants of the R2 generation. The resistance to PVYN and PVYO strains was maintained when the plants were inoculated dually with the potato potyviruses V or A (PVV or PVA). Only partial resistance to PVYN 605 was observed in plants containing the CP coding sequence in an antisense orientation. Both the "sense" and "antisense" plants accumulated CP transcripts and possessed multiple copies of the transgene. Systemic spread of the virus was studied by grafting experiments. The transgenic PVYN 605 CP was undetectable in healthy plants. This protein accumulated in plants of PVY-susceptible CP-transgenic line 4B5-31 when infected by PVYO, PVA, or PVV, but not in plants of line 4B5-S4, that were resistant to PVY and tolerant to PVA or PVV. To assess the role of the transgenic PVYN 605 CP in protection, we transformed plants with a construct bearing a frame-shift mutation at the beginning of the CP coding sequence, and observed partial protection from PVYN 605. Several hypotheses to explain these resistance mechanisms are discussed.

Potato plants transgenic for the coat protein (CP) gene of Potato virus Y strain N (PVY(N)) were examined for the accumulation of the transgenic PVY(N) CP. The protein remained undetectable in healthy plants of lines Bt6 and Bt10 that are completely or partially resistant to this virus, but accumulated when the plants were infected by a related strain, PVY(O). One possible explanation was that the transgenic CP is stabilized by integration into the capsid of the infecting virus. To investigate this, virus particles originating from these plants were examined by immuno-electron microscopy. Heterologous encapsidation was observed between the infecting PVY(O) virus and the transgenic PVY(N) CP. These results have implications for the understanding of the mechanism of CP-mediated protection and assessing its impact on agricultural ecosystems.

Tostudy theimport ofpolypeptide precursors wehaveadapted andcompared twoprocedures fortheisolation ofcompetent chloroplasts fromthegreenunicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation andelutriation. The chloroplasts actively import theprecursor ofthesmall subunit of ribulose bisphosphate carboxylase-oxygenase invitro. press atlowpressure (16). Intact chloroplasts canthusbe recovered whicharecompetent fortheinvitro import of radiolabeled precursor polypeptides (3, 4). Wehavecompared twoapproaches fortheir isolation: silicasol gradient centrifu- gation andelutriation (19, 24).

To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro.

Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.

D1 and D2, two chloroplast proteins with apparent mol. wt of 32 000‐34 000, play an important role in the photosynthetic reactions mediated by the membrane‐bound protein complex of photosystem II (PSII). We have isolated and characterized an uniparental, non‐photosynthetic mutant of Chlamydomonas reinhardtii and show that the mutation is in the chloroplast gene psbD, coding for D2. A 46 bp direct DNA duplication in the coding region of the mutant gene causes a frame‐shift which results in a psbD transcript coding for 186 amino acid residues instead of the normal 352. The truncated D2 peptide is never seen, even after pulse‐labeling, suggesting that the mutant protein is very unstable. In addition, little or no D1 protein is detected in this mutant although the gene and normal levels of mRNA for D1 are present in mutant cells. All other core PSII proteins are synthesized and inserted into the membrane fraction, but never accumulate. These results suggest that D2 contributes not only to the stabilization of the PSII complex in the membrane, but also may play a specific role in the regulation of the D1 protein, either at the translational or post‐translational level.

The region of the chloroplast genome of Chlamydomonas reinhardii containing the gene of the thylakoid polypeptide D2 (psbD) has been sequenced. A unique open reading frame of 350 codons exists in this region. Because the first ATG is followed 11 codons downstream by a second one, the D2 polypeptide consists of either 339 or 350 amino acids. Comparison of the sequences of D2 and the 32K dalton polypeptides, both of which are associated with photosystem II, reveals partial homology. Although, the overall homology of these two polypeptides is only 27%, they contain several related regions and their hydropathic profiles are strikingly similar. These data suggest that the two polypeptides may have related functions and/or that their genes may have originated from a common ancestor. Alternatively, convergent evolution of these polypeptides may be due to structural constraints in the thylakoid membrane. Limited sequence homology is also observed between the D2 polypeptide and some of the subunits of the reaction centers of photosynthetic bacteria.

The gene coding for the large subunit of ribulose 1,5-diphosphate carboxylase (LS) of Chlamydomonas reinhardii has been cloned and localized at a unique site on the physical map of the chloroplast genome. It is contained within a 5.5 × 103 base EcoRI fragment which can act as template in vitro in a coupled transcription-translation system to produce a polypeptide, of the same size as LS, which is specifically immunoprecipitated with antibodies prepared against LS. The fingerprint of this in vitro synthesized polypeptide closely resembles that of cellular LS. The hybrid plasmid coding for LS inhibits specifically the in vitro translation of LS when it is hybridized to poly(A)− RNA prior to translation. The size of the LS messenger RNA has been estimated at 1.2 ± 0.3 × 103 bases by measuring R-loop structures in the electron microscope and two major species at 1.5 × 103 and 2.5 × 103 bases have been detected by hybridizing labelled LS-plasmid to cellular RNA fractionated on denaturing agarose gels.

Two ribosomal DNA (rDNA) regions exist in the circular chloroplast genome of C. reinhardii. They are located at widely distant sites on the map in an inverted orientation. The chloroplast rDNA has been cloned and a fine structure map has been established. The small chloroplast ribosomal subunit contains 16S RNA, while the large subunit contains the four distinct RNA species 23, 7, 5 and 3S rRNA. The location of the rRNA genes has been determined by electron microscopy and by hybridization of the purified rRNAs to various rDNA restriction fragments. The gene order in the direction of transcription is 16, 7, 3, 23 and 5S. A spacer (Sp) of 1.68 ± 0.13 kb separates the 16S and the 7S RNA genes, and a very small spacer of 0.16 ± 0.04 kb separates the 7S and 23S RNA genes. The 5, 23, 3 and 7S RNAs are transcribed on the same strand.