Nobuhiro Kita

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, α-D-GalpA-([1→4]-α-D-GalpA)3-(1→4)-D-GalpA, is composed of five galacturonopyranose units (D-GalpA) linked by α-(1→4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein–oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC–galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the β elimination cleavage of polygalacturonic acid.

Development of biological soil disinfestations in Japan

Biological soil disinfestations (BSDs) were developed separately in Japan and in The Netherlands as an alternative to chemical fumigations. In Japan, it was developed based on the knowledge of irrigated paddy rice and upland crop rotation system that was rather tolerant of soil-borne disease development. The methods consist of application of easily decomposable organic matter, irrigation, and covering the soil surface with plastic film, thereby inducing anaerobic (reductive) soil conditions and suppressing many soil-borne pests including fungi, bacteria, nematodes, and weeds. The methods are widely used by organic farmers in the area where residences and agricultural fields are intermingled. To note one advantage of these methods, maintenance of soil suppressiveness to Fusarium wilt of tomato was suggested, while soil treated with chloropicrin became conducive to the disease. Suppression of soil-borne fungal pathogens by BSDs might be attributed to anaerobicity and high temperature, organic acids generated, and metal ions released into soil water. Contributions of respective factors to suppression of respective pathogens might be diverse. Presumably, these factors might vary on the fungal community structure in BSD-treated soil. These factors also work in paddy fields. Therefore, the BSDs developed in Japan are probably a method to raise the efficacy of paddy–upland rotation through intensive organic matter application and through maintenance of a strongly anaerobic (reductive) soil condition.

Selection of bacterial wilt-resistant tomato through tissue culture.

Bacterial wilt-resistant plants were obtained using a tomato tissue culture system. A virulent strain ofPseudomonas solanacearum secreted some toxic substances into the culture medium. Leaf explant-derived callus tissues which were resistant to these toxic substances in the culture filtrate were selectedin vitro and regenerated into plants. These plants expressed bacterial wilt resistance at the early infection stage to suppress or delay the growth of the inoculated bacteria. On the other hand, complete resistance was obtained in self-pollinated progeny of regenerants derived from non-selected callus tissues. These plants showed a high resistance when inoculated with this strain, and were also resistant when planted in a field infested with a different strain of the pathogen.

Functional implications of the three-dimensional structures of pectate lyases

Abstract The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, PelC and PelE, have been refined to a resolution of 2.2. A superposition of the two structures has been used to correct the multiple sequence alignment of the extracellular pectate lyase superfamily. The corrected alignment has revealed two clusters of ‘potentially catalytic’ invariant amino acids, compatible with two active sites. Site-directed mutagenesis studies have confirmed that the pectinolytic active site includes the region around the Ca2+ binding site. Furthermore, mutagenesis studies suggest catalytic roles for individual amino acids. Although PelC and PelE are structurally similar in the overall fold of the polypeptide backbone, there are significant differences in the size and conformation of the polypeptide backbone, there are significant differences in the size and conformation of the loops that comprise the pectinolytic active site. The differences in the surface charges between PelC and PelE in the groove extending from the Ca2+ site suggest that the optimal oligosaccharide substrates are different for each enzyme.

Suppression of transgene RNA silencing in transgenic Arabidopsis thaliana by a mild strain of Cucumber mosaic virus

The ability of Cucumber mosaic virus (CMV) subgroup II strain CMV-KT to suppress RNA silencing via posttranscriptional gene silencing (PTGS) was determined using Turnip mosaic virus (TuMV)-resistant transgenic Arabidopsis thaliana (TuR-At) carrying TuMV coat protein (CP) transgenes. CMV-KT induced only slight growth retardation in TuR-At plants, but no dwarf or mosaic symptoms, while subgroup I strain Y (CMV-Y) caused severe symptoms. Expression of the RNA-silenced TuMV-CP transgene resumed and produced TuMV-CP in either CMV-KT- or CMV-Y-infected TuR-At plants, indicating that the RNA silencing of the TuMV-CP gene was suppressed. Challenge inoculation experiments revealed that RNA silencing of the TuMV-CP transgene by CMV-KT infection is suppressed mostly in young developing leaves, but not in mature leaves, of TuR-At plants. These results demonstrated that transgene-derived virus resistance in transgenic Arabidopsis plants can be compromised by even a mild strain of CMV infection.