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Plant defense involves a complex array of biochemical interactions, many of which occur in the extracellular environment. The apical 1- to 2-mm root tip housing apical and root cap meristems is resistant to infection by most pathogens, so growth and gravity sensing often proceed normally even when other sites on the root are invaded. The mechanism of this resistance is unknown but appears to involve a mucilaginous matrix or \"slime\" composed of proteins, polysaccharides, and detached living cells called \"border cells.\" Here, we report that extracellular DNA (exDNA) is a component of root cap slime and that exDNA degradation during inoculation by a fungal pathogen results in loss of root tip resistance to infection. Most root tips (>95%) escape infection even when immersed in inoculum from the root-rotting pathogen Nectria haematococca. By contrast, 100% of inoculated root tips treated with DNase I developed necrosis. Treatment with BAL31, an exonuclease that digests DNA more slowly than DNase I, also resulted in increased root tip infection, but the onset of infection was delayed. Control root tips or fungal spores treated with nuclease alone exhibited normal morphology and growth. Pea (Pisum sativum) root tips incubated with [³²P]dCTP during a 1-h period when no cell death occurs yielded root cap slime containing ³²P-labeled exDNA. Our results suggest that exDNA is a previously unrecognized component of plant defense, an observation that is in accordance with the recent discovery that exDNA from white blood cells plays a key role in the vertebrate immune response against microbial pathogens.

To our best knowledge, all of the fungal immunomodulatory proteins (FIPs) have been successfully extracted and identified in Basidomycetes, with only the exception of FIP from ascomycete Nectria haematococca (FIP-nha) discovered through homology alignment most recently. In this work, a gene encoding FIP-nha was synthesized and recombinantly expressed in an Escherichia coli expression system. SDS-PAGE and MALDI-MS analyses of recombinant FIP-nha (rFIP-nha) indicated that the gene was successfully expressed. The yield of the bioactive FIP-nha protein was 42.7 mg/L. In vitro assays of biological activity indicated that the rFIP-nha caused hemagglutination of human and rabbit red blood cells, significantly stimulated mouse spleen lymphocyte proliferation, and enhanced expression of interleukin-2 (IL-2) released from mouse splenocytes, revealing a strong antitumor effect against HL60, HepG2 and MGC823. Through this work, we constructed a rapid and efficient method of FIP production, and suggested that FIP-nha is a valuable candidate for use in future medical care and pharmaceutical products.

Cosmospora sensu Rossman accommodated nectroid fungi with small, reddish, smooth, thin-walled perithecia but recently was found to be polyphyletic and has been segregated into multiple genera. Not all cosmospora-like fungi have been treated systematically. Some of these species include C. vilior and many specimens often labeled \"Cosmospora sp.\" The objectives of this research were to establish the identity of C. vilior through epitypication using a recent collection that agrees with the type specimen in morphology, host and geography and to determine its phylogenetic position within Cosmospora sensu lato and the Nectriaceae. A multilocus phylogeny was constructed based on six loci (ITS, LSU, MCM7, rpb1, tef1, tub) to estimate a phylogeny. Results from the phylogenetic analyses indicated that C. vilior forms a monophyletic group with other cosmospora-like fungi that have an acremonium-like anamorph and that parasitize Eutypa and Eutypella (Ascomycota, Sordariomycetes, Xylariales, Diatrypaceae). The group is phylogenetically distinct from other previously segregated genera. A new genus, Pseudocosmospora, is described to accommodate the type species, P. eutypellae, and nine additional species in this clade.

In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.

Karyotypes of the cucurbit pathogen Nectria haematococca MPI (anamorph Fusarium solani f. sp. cucurbitae race 1) was studied using the two standard strains ATCC18098 and ATCC18099. Complete separation of all chromosomes was difficult with pulsed field gel electrophoresis due to both the large size and co-migration of chromosomes. In contrast, cytological karyotyping was done successfully with fluorescence microscopy combined with the germ tube burst method for sample preparation to visualize mitotic metaphase chromosomes. For each strain the basic chromosome number (CN) was nine, which revises previous chromosome estimates of n = 4. Chromosomes were morphologically characterized by their sizes, intensely fluorescing segments, and protrusion of rDNA. In addition to the basic chromosome complement, ATCC18098 had a mini-chromosome of ~410 kb present as a single copy in somatic nuclei. Chromosome fluorescence in situ hybridization indicated that this mini-chromosome is not a derivative from the other chromosomes in the genome. In addition, crossing experiments suggested that it was transmitted in a Mendelian manner to the ascospore progeny.

Root infection in susceptible host species is initiated predominantly in the zone of elongation, whereas the remainder of the root is resistant. Nectria haematococca infection of pea (Pisum sativum) was used as a model to explore possible mechanisms influencing the localization of root infection. The failure to infect the root tip was not due to a failure to induce spore germination at this site, suppression of pathogenicity genes in the fungus, or increased expression of plant defense genes. Instead, exudates from the root tip induce rapid spore germination by a pathway that is independent of nutrient-induced germination. Subsequently, a factor produced during fungal infection and death of border cells at the root apex appears to selectively suppress fungal growth and prevent sporulation. Host-specific mantle formation in response to border cells appears to represent a previously unrecognized form of host-parasite relationship common to diverse species. The dynamics of signal exchange leading to mantle development may play a key role in fostering plant health, by protecting root meristems from pathogenic invasion.

Most infections of plant roots are initiated in the region of elongation; the mechanism for this tissue-specific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead of 24 h, and border cell number is decreased by more than 99%. These plants were found to exhibit increased root-tip infection by a fungal pathogen and reduced nodule formation by a bacterial symbiont. Thus, the frequency of infection in the region of elongation by Nectria haematocca was unaffected, but infection of the root tip was increased by more than 90%; early stages of Sinorhizobium meliloti infection and nodule morphology were normal, but the frequency of nodulation was fourfold lower than in wild-type roots.

The karyotypes of three isolates of Mycosphaerella graminicola, the septoria tritici blotch pathogen of wheat, were analyzed with both pulsed field gel electrophoresis (PFGE) and the cytological technique called germ tube burst method (GTBM). These analyses revealed a chromosome length polymorphism among these isolates. The estimated genome size was 31-40 Mb depending on the isolates, indicating 17-22% redundancy in the genome of the standard strain IPO323 because such differences do not affect development, pathogenicity and sexual reproduction of the other isolates. The chromosome numbers in the three isolates were 18-20 and the chromosome size was 0.3-6 Mb. These data show that M. graminicola has the highest chromosome number and the smallest autosomes (A chromosomes) in filamentous ascomycetes. Our data also confirmed a large (≥ 6 Mb) chromosome that was assembled recently in the IPO323 genome sequence. GTBM analyses revealed the mitotic metaphase chromosomes, enabling chromosome quantification, which was fully congruent with the PFGE analyses. These data will be instrumental in the final assembly of the M. graminicola genome.

Previous studies identified a cluster of six genes that are expressed in the fungus Nectria haematococca mating population VI during infection of pea. Four of these genes were shown to contribute to pathogenicity on pea and were called PEP genes for pea pathogenicity. The cluster is located on a \"conditionally dispensable\" (CD) chromosome and has features similar to bacterial pathogenicity islands. In this study, the occurrence and location of members of the PEP cluster were analyzed in laboratory strains and nine pea pathogenic and 16 non-pea pathogenic field isolates of N. haematococca. Our results indicate that all pea-pathogenic isolates have homologues for all six genes present in the PEP cluster and the homologues appear to be clustered. PEP homologues are also present in isolates that are not pathogenic on pea, although none of these isolates have homologues of all six genes. In addition, PEP homologues are found in CD chromosomes and in other chromosomes. Isolates without PEP homologues are virulent on ripe tomato fruits and carrot roots, indicating that PEP genes are not required for pathogenicity on these hosts.

A new genus in the Hypocreales, Leuconectria, is described for Pseudonectria clusiae based on its unique perithecial wall anatomy and the anamorph Gliocephalotrichum bulbilium produced from single ascospores of L. clusiae. The teleomorph had previously been placed in Pseudonectria, a genus defined for Nectria-like species with nonseptate ascospores. Pseudonectria is redefined based on the type species P. rousseliana and its anamorph Volutella buxi. Pseudonectria rousseliana causes a disease of boxwood (Buxus sempervirens) in the Buxaceae. The two other species included in Pseudonectria, P. coronata and P. pachysandricola, also occur on members of the Buxaceae. All three species are described and illustrated, and a key to species is provided.