Bark and wood tissues of American elm exhibit distinct responses to Dutch elm disease.

Tolerance to Dutch elm disease (DED) has been linked to the rapid and/or high induction of disease-responsive genes after infection with the fungus Ophiostoma novo-ulmi. Although the fungal infection by O. novo-ulmi primarily takes places in xylem vessels, it is still unclear how xylem contributes to the defense against DED. Taking advantage of the easy separation of wood and bark tissues in young American elm saplings, here we show that most disease-responsive genes exhibited higher expression in wood compared to bark tissues after fungal infection. On the other hand, the stress-related phytohormones were generally more abundant in the bark compared to wood tissues. However, only endogenous levels of jasmonates (JAs), but not salicylic acid (SA) and abscisic acid (ABA) increased in the inoculated tissues. This, along with the upregulation of JA-biosynthesis genes in inoculated bark and core tissues further suggest that phloem and xylem might contribute to the de novo biosynthesis of JA after fungal infection. The comparison between two tolerant elm varieties, 'Valley Forge' and 'Princeton,' also indicated that tolerance against DED might be mediated by different mechanisms in the xylem. The present study sheds some light on the amplitude and kinetics of defense responses produced in the xylem and phloem in response to DED.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease.

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions.

Protoplast-to-plant regeneration of American elm (Ulmus americana).

This study describes a protocol for regeneration of plants from cell suspension-derived protoplasts of American elm (Ulmus americana). Efficient protoplast isolation was achieved from a two-phase culture system through the incorporation of 100 µM 2-aminoindan-2-phosphonic acid, with a yield of approximately 2 × 10(6) protoplasts/ml packed cell volume. Isolated protoplasts failed to survive in liquid or alginate bead culture systems but initiated and continued to divide when embedded in low melting point agarose beads. Protoplast-derived callus proliferated and differentiated into shoot buds in response to 10 or 20 µM thidiazuron. Differentiated buds elongated and continued to proliferate on elm shoot medium supplemented with 3.0 µM GA3. The protoplast-derived shoots rooted and acclimatized to greenhouse conditions and continued to grow. This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protoplast fusion or genome editing technologies.

Establishment of invasive and non-invasive reporter systems to investigate American elm-Ophiostoma novo-ulmi interactions.

Dutch elm disease (DED), caused by ascomycete fungi in the Ophiostoma genus, is the most devastating disease of American elm (Ulmus americana) trees. Cerato ulmin (CU), a hydrophobin secreted by the fungus, has been implicated in the development of DED, but its role in fungal pathogenicity and virulence remains uncertain and controversial. Here, we describe reporter systems based on the CU promoter and three reporter proteins (GFP, GUS and LUC), developed as research tools for quantitative and qualitative studies of DED in vitro, in vivo and in planta. A strain of the aggressive species Ophiostoma novo-ulmi was transformed with the reporter constructs using Agrobacterium-mediated transformation and the fungal transformants, namely M75-GFP, M75-GUS and M75-LUC, were examined for mitotic stability after repeated subcultures. The intensity of GFP fluorescence was strong in M75-GFP spores and hyphae, allowing microscopic investigations of spore structure, fungal morphogenesis and fungal development. The interaction of M75-GFP and U. americana callus cells was explored with scanning laser confocal microscopy facilitating qualitative studies on fungal strategies for the invasion and penetration of elm cells. M75-GUS was generated to provide an invasive, yet quantitative approach to study fungal-plant interactions in vitro and in planta. The generation of M75-LUC transformants was aimed at providing a non-destructive quantitative approach to study the role of CU in vivo. The sensitivity, low background signal and linearity of LUC assays all predict a very reliable approach to investigate and re-test previously claimed roles of this CU in fungal pathogenicity. These reporter systems provide new tools to investigate plant-pathogen interactions in this complex pathosystem and may aid in better understanding the development of DED.