Evidence of Coevolution Between Cronartium harknessii Lineages and Their Corresponding Hosts, Lodgepole Pine and Jack Pine.

Variation in rate of infection and susceptibility of Pinus spp. to the fungus Cronartium harknessii (syn. Endocronartium harknessii), the causative agent of western gall rust, has been well documented. To test the hypothesis that there is a coevolutionary relationship between C. harknessii and its hosts, we examined genetic structure and virulence of C. harknessii associated with lodgepole pine (P. contorta var. latifolia), jack pine (P. banksiana), and their hybrids. A secondary objective was to improve assessment and diagnosis of infection in hosts. Using 18 microsatellites, we assessed genetic structure of C. harknessii from 90 sites within the ranges of lodgepole pine and jack pine. We identified two lineages (East and West, FST = 0.677) associated with host genetic structure (r = 0.81, P = 0.001), with East comprising three sublineages. In parallel, we conducted a factorial experiment in which lodgepole pine, jack pine, and hybrid seedlings were inoculated with spores from the two primary genetic lineages. With this experiment, we refined the phenotypic categories associated with infection and demonstrated that stem width can be used as a quantitative measure of host response to infection. Overall, each host responded differentially to the fungal lineages, with jack pine exhibiting more resiliency to infection than lodgepole pine and hybrids exhibiting intermediate resiliency. Taken together, the shared genetic structure between fungus and host species, and the differential interaction of the fungal species with the hosts, supports a coevolutionary relationship between host and pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A novel application of RNase H2-dependent quantitative PCR for detection and quantification of Grosmannia clavigera, a mountain pine beetle fungal symbiont, in environmental samples.

Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry). These fungi are vectored by MPB to new pine hosts, where the fungi overcome host defenses to grow into the sapwood. A tree's relative susceptibility to these fungi is conventionally assessed by measuring lesions that develop in response to fungal inoculation. However, these lesions represent a symptom of infection, representing both fungal growth and tree defense capacity. In order to more objectively assess fungal virulence and host tree susceptibility in studies of host-pathogen interactions, a reliable, consistent, sensitive method is required to accurately identify and quantify MPB-associated fungal symbionts in planta. We have adapted RNase H2-dependent PCR, a technique originally designed for rare allele discrimination, to develop a novel RNase H2-dependent quantitative PCR (rh-qPCR) assay that shows greater specificity and sensitivity than previously published PCR-based methods to quantify MPB fungal symbionts in pine xylem and MPB whole beetles. Two sets of assay probes were designed: one that amplifies a broad range of ophiostomoid species, and a second that amplifies G. clavigera but not other MPB-associated ophiostomoid species. Using these primers to quantify G. clavigera in pine stems, we provide evidence that lesion length does not accurately reflect the extent of fungal colonization along the stem nor the quantity of fungal growth within this colonized portion of stem. The sensitivity, specificity, reproducibility, cost effectiveness and high-throughput potential of the rh-qPCR assay makes the technology suitable for identification and quantification of a wide array of pathogenic and beneficial microbes that form associations with plants and other organisms, even when the microbial partner is present in low abundance.

Alanine aminotransferase variants conferring diverse NUE phenotypes in Arabidopsis thaliana.

Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.

Analysis of the enzymatic properties of a broad family of alanine aminotransferases.

Alanine aminotransferase (AlaAT) has been studied in a variety of organisms due to the involvement of this enzyme in mammalian processes such as non-alcoholic hepatocellular damage, and in plant processes such as C4 photosynthesis, post-hypoxic stress response and nitrogen use efficiency. To date, very few studies have made direct comparisons of AlaAT enzymes and fewer still have made direct comparisons of this enzyme across a broad spectrum of organisms. In this study we present a direct kinetic comparison of glutamate:pyruvate aminotransferase (GPAT) activity for seven AlaATs and two glutamate:glyoxylate aminotransferases (GGAT), measuring the K(M) values for the enzymes analyzed. We also demonstrate that recombinant expression of AlaAT enzymes in Eschericia coli results in differences in bacterial growth inhibition, supporting previous reports of AlaAT possessing bactericidal properties, attributed to lipopolysaccharide endotoxin recognition and binding. A probable lipopolysaccharide binding region within the AlaAT enzymes, homologous to a region of a lipopolysaccharide binding protein (LBP) in humans, was also identified in this study. The AlaAT enzyme differences identified here indicate that AlaAT homologues have differentiated significantly and the roles these homologues play in vivo may also have diverged significantly. Specifically, the differing kinetics of AlaAT enzymes and how this may alter the nitrogen use efficiency in plants is discussed.

Engineering nitrogen use efficient crop plants: the current status.

In the last 40 years the amount of synthetic nitrogen (N) applied to crops has risen drastically, resulting in significant increases in yield but with considerable impacts on the environment. A requirement for crops that require decreased N fertilizer levels has been recognized in the call for a 'Second Green Revolution' and research in the field of nitrogen use efficiency (NUE) has continued to grow. This has prompted a search to identify genes that improve the NUE of crop plants, with candidate NUE genes existing in pathways relating to N uptake, assimilation, amino acid biosynthesis, C/N storage and metabolism, signalling and regulation of N metabolism and translocation, remobilization and senescence. Herein is a review of the approaches taken to determine possible NUE candidate genes, an overview of experimental study of these genes as effectors of NUE in both cereal and non-cereal plants and the processes of commercialization of enhanced NUE crop plants. Patents issued regarding increased NUE in plants as well as gene pyramiding studies are also discussed as well as future directions of NUE research.