Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Molecular cloning and analysis of apple HcrVf resistance gene paralogs in a collection of related Malus species

Few complete genes belonging to the receptor-like protein class of plant resistance (R) genes (called HcrVf genes in Malus) have been cloned from apple cultivars. To date, the HcrVf2 gene from the Rvi6 locus of ??Florina??, a derivative of Malus?×?floribunda 821, is the only cloned apple scab R gene with a proven function. The breakdown of the Rvi6 scab resistance in several apple growing regions has forced the search for new resistance sources for R gene pyramiding through traditional and biotechnological breeding. Marker-assisted breeding is aimed at the selection of the desired R gene combinations but might be extended for monitoring putative risks of resistance breakdown in potential scab R gene donors. Here we report on a marker-based screen of Rvi6 homologues supplemented by a polymerase chain reaction (PCR)-based full-length cloning of HcrVf paralogs. Known Rvi6 markers were analysed in a sub-set of accessions selected by a preceding SSR-based genetic relationship analysis from a large Malus species germplasm collection, which has been evaluated for scab resistance in an unsprayed orchard for a period of 3?years. The Rvi6 breakdown in several M. × floribunda accessions was confirmed, and several other Malus species putatively related to M. × floribunda were also infected by scab. The selected sub-cluster consisting of 40 accessions, including all M. × floribunda, two Malus?×?micromalus and two Malus baccata accessions, was screened for Rvi6 markers CH-Vf1-SSR and AL07-SCAR and for the presence of HcrVf2 by using gene-specific primers. The two M. × micromalus accessions, which proved to be identical genotypes, were found to be closely related to M. × floribunda. They also displayed the Rvi6 markers and could be infected by race (5,6,7) scab isolate Vi158. To verify the assumed existence of the HcrVf2 gene in M. × micromalus, a PCR-based cloning method was used to clone full-length HcrVf paralogs from this species and additionally from a scab-susceptible M. baccata genotype also showing the Rvi6 markers. The M. × micromalus gene MAM31 was identified as an identical copy of HcrVf2. Another HcrVf-like gene (MAM6) newly cloned from M. × micromalus showed 95 % similarity to HcrVf2. MAM6 was chosen for the development of a gene-specific PCR marker, which was analysed in the selected apple group and additionally mapped in an apple progeny derived from a cross with M. × micromalus. The cloning method described in this paper might be used in future to mine for more HcrVf gene variants to develop highly specific markers for R gene deployment in traditional breeding and to use cloned genes for gene transfer and functional studies.     

Identification of pseudouridine methyltransferase in Escherichia coli

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m³Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m³Ψ1915 is the only methylated pseudouridine in bacteria described to date.     

Ribosome assembly in Escherichia coli strains lacking the RNA helicase DeaD/CsdA or DbpA

Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 degrees C and at 37 degrees C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD(-) strain. Pulse labeling showed that the 40S particles are converted nearly completely into functional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD-deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD-catalyzed structural transitions of the ribosome large subunit assembly.     

Kinetic Constants for Aerobic Growth of Microbial Populations Selected with Various Single Compounds and with Municipal Wastes as Substrates

The general applicability of the Monod relationship between the logarithmic growth rate constant and substrate concentration was studied for heterogeneous populations metabolizing a variety of substrates including concentrated municipal sewage. It was found that growth could be described by the Monod equation, mu = mu(m)/k(s) + s. The kinetic "constants" for heterogeneous populations growing on concentrated sewage were comparable to those found with glucose as substrate.     

The determination of the parameter of Bertalanffy's growth differential equations by means of nonlinear internal regression

A short survey is given on various parameterized versions of the logistic law of growth and of Bertalanffy's growth differential equations. To examine the validity of these various growth expressions internal nonlinear regressions were performed, and the results of the calculations are presented. The body length growth of man within the embryonic development serves as examples of a growth process. The parameters in the differential equations will be adjusted to the course of the divided central differences calculated from means of measured values of this growth process.     

Quantitative mathematical evaluation of growth studies from the viewpoint of exogenously or genetically influenced variability

Firstly, the ideas are sketched which serve as the basis for the phenomenologic-mathematical kind of modeling of the body length growth process of man. For proving the biological relevance of the spurts analyzed by numerical procedures one has to consider the social and the biological circumstances in which the growth process takes place. An analysis of longitudinal data series of the body length of (monozygotic) twins will give further hints to the possible meaning of the growth spurts by way of separation of exogenous and of genetic determined endogenous agents on the growth process. The data available for our examinations cover the time interval of the praepubertal and the postpuberal development as well as the time interval in which the puberal growth spurt takes place. The way to proceed in evaluation these time series will be presented.     

Phenomenologic-mathematical modeling of the human-body-length growth through the analysis of growth periods and their quantitative-analytical registration

The body height growth (of masculine beings) was modelled in a phenomenologic-mathematical manner by partitioning the time course of measured growth curve in parts every of which corresponds to a separated growth period. This partitioning was reached in a natural way so that a superposition of the single spurts yields the whole measured course. Every growth batch will be described in its time course by one term of inverse tangent function. The biological meaning and an explanation of the succession of the growth spurt as an effect of control circuits need further exploratory work. For detailed statements on acceleration phenomena concerning the body height growth this analysis gives possibilities for comparing the single growth spurts of the mean growth process of two populations in question. For measured values given by BROCK (1954) and SALZLER (1967) there are five growth periods in the time intervall reaching from time of conception until the end of the first year. Comparing the mathematical functions of the corresponding growth spurts for these two groups one can conclude that the second spurt (fetal spurt) is responsible for an increase of birth body height and the fourth for an increase of body height in the suckling age of the latter group against the former one.     

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight

Planta - Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were...     

Nonparametric estimation of 1-dimensional continuous distribution density functions using the continuous LOLINREG approximation

A nonparametric method for estimation of one-dimensional continuous probability distribution functions is presented. Procedures for calculation of estimation of the unknown distribution function and the distribution density will be discussed in their application. 2 items are what type of weight function may be chosen for the proposed local-linear continuous approximation of the empirical distribution function by the least squares method (LOLINREG), and upon what value of bandwidth- or smoothing parameter one optimally should settle. The latter problem is practically very important with respect to the quality of the estimation results. Examples of simulated measurements which come from a standardized normal distribution as random numbers serve to demonstrate the mode of working, the advantages as well as the limits of the presented continuous LOLINREG-approximation.