Justin G. A. Whitehill

The U-Box/ARM E3 Ligase PUB13 Regulates Cell Death, Defense, and Flowering Time in Arabidopsis

The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

Distinguishing defensive characteristics in the phloem of ash species resistant and susceptible to emerald ash borer.

We examined the extent to which three Fraxinus cultivars and a wild population that vary in their resistance to Emerald Ash Borer (EAB) could be differentiated on the basis of a suite of constitutive chemical defense traits in phloem extracts. The EAB-resistant Manchurian ash (F. mandshurica, cv. Mancana) was characterized by having a rapid rate of wound browning, a high soluble protein concentration, low trypsin inhibitor activities, and intermediate levels of peroxidase activity and total soluble phenolic concentration. The EAB-susceptible white ash (F. americana, cv. Autumn Purple) was characterized by a slow wound browning rate and low levels of peroxidase activity and total soluble phenolic concentrations. An EAB-susceptible green ash cultivar (F. pennsylvanica, cv. Patmore) and a wild accession were similar to each other on the basis of several chemical defense traits, and were characterized by high activities of peroxidase and trypsin inhibitor, a high total soluble phenolic concentration, and an intermediate rate of wound browning. Lignin concentration and polyphenol oxidase activities did not differentiate resistant and susceptible species. Of 33 phenolic compounds separated by HPLC and meeting a minimum criterion for analysis, nine were unique to Manchurian ash, five were shared among all species, and four were found in North American ashes and not in the Manchurian ash. Principal components analysis revealed clear separations between Manchurian, white, and green ashes on the basis of all phenolics, as well as clear separations on the basis of quantities of phenolics that all species shared. Variation in some of these constitutive chemical defense traits may contribute to variation in resistance to EAB in these species.

Interspecific Comparison of Constitutive Ash Phloem Phenolic Chemistry Reveals Compounds Unique to Manchurian Ash, a Species Resistant to Emerald Ash Borer

The emerald ash borer (Agrilus planipennis, EAB) is an invasive wood-borer indigenous to Asia and is responsible for widespread ash (Fraxinus spp.) mortality in the U.S. and Canada. Resistance and susceptibility to EAB varies among Fraxinus spp., which is a result of their co-evolutionary history with the pest. We characterized constitutive phenolic profiles and lignin levels in the phloem of green, white, black, blue, European, and Manchurian ash. Phloem was sampled twice during the growing season, coinciding with phenology of early and late instar EAB. We identified 66 metabolites that displayed a pattern of variation, which corresponded strongly with phylogeny. Previously identified lignans and lignan derivatives were confirmed to be unique to Manchurian ash, and may contribute to its high level of resistance to EAB. Other compounds that had been considered unique to Manchurian ash, including hydroxycoumarins and the phenylethanoids calceolarioside A and B, were detected in closely related, but susceptible species, and thus are unlikely to contribute to EAB resistance of Manchurian ash. The distinct phenolic profile of blue ash may contribute to its relatively high resistance to EAB.

Effects of water availability on emerald ash borer larval performance and phloem phenolics of Manchurian and black ash

The invasive emerald ash borer (EAB) beetle is a significant threat to the survival of North American ash. In previous work, we identified putative biochemical and molecular markers of constitutive EAB resistance in Manchurian ash, an Asian species co-evolved with EAB. Here, we employed high-throughput high-performance liquid chromatography with photodiode array detection and mass spectrometry (HPLC-PDA-MS) to characterize the induced response of soluble phloem phenolics to EAB attack in resistant Manchurian and susceptible black ash under conditions of either normal or low water availability, and the effects of water availability on larval performance. Total larval mass per tree was lower in Manchurian than in black ash. Low water increased larval numbers and mean larval mass overall, but more so in Manchurian ash. Low water did not affect levels of phenolics in either host species, but six phenolics decreased in response to EAB. In both ashes, pinoresinol A was induced by EAB, especially in Manchurian ash. Pinoresinol A and pinoresinol B were negatively correlated with each other in both species. The higher accumulation of pinoresinol A in Manchurian ash after attack may help explain the resistance of this species to EAB, but none of the responses measured here could explain increased larval performance in trees subjected to low water availability.

Interspecific proteomic comparisons reveal ash phloem genes potentially involved in constitutive resistance to the emerald ash borer.

The emerald ash borer (Agrilus planipennis) is an invasive wood-boring beetle that has killed millions of ash trees since its accidental introduction to North America. All North American ash species (Fraxinus spp.) that emerald ash borer has encountered so far are susceptible, while an Asian species, Manchurian ash (F. mandshurica), which shares an evolutionary history with emerald ash borer, is resistant. Phylogenetic evidence places North American black ash (F. nigra) and Manchurian ash in the same clade and section, yet black ash is highly susceptible to the emerald ash borer. This contrast provides an opportunity to compare the genetic traits of the two species and identify those with a potential role in defense/resistance. We used Difference Gel Electrophoresis (DIGE) to compare the phloem proteomes of resistant Manchurian to susceptible black, green, and white ash. Differentially expressed proteins associated with the resistant Manchurian ash when compared to the susceptible ash species were identified using nano-LC-MS/MS and putative identities assigned. Proteomic differences were strongly associated with the phylogenetic relationships among the four species. Proteins identified in Manchurian ash potentially associated with its resistance to emerald ash borer include a PR-10 protein, an aspartic protease, a phenylcoumaran benzylic ether reductase (PCBER), and a thylakoid-bound ascorbate peroxidase. Discovery of resistance-related proteins in Asian species will inform approaches in which resistance genes can be introgressed into North American ash species. The generation of resistant North American ash genotypes can be used in forest ecosystem restoration and urban plantings following the wake of the emerald ash borer invasion.

Ips pini (Curculionidae: Scolytinae) Is a Vector of the Fungal Pathogen, Sphaeropsis sapinea (Coelomycetes), to Austrian Pines, Pinus nigra (Pinaceae)

Abstract Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton, is among the most common and widely distributed pathogens of conifers worldwide. S. sapinea is disseminated over short distances by rain splash and moist wind, but significant knowledge gaps regarding long-range dispersal remain. Our objective was to determine whether or not the pine engraver beetle, Ips pini Say, is a vector of the pathogen onto Austrian pines (Pinus nigra Arnold). In 2004 and 2005, individuals of I. pini were collected with pheromone traps at two locations in central Ohio (197 and 1,017 individuals for 2004 and 2005, respectively) and screened for the presence of S. sapinea. In the field, fresh logs of Austrian pine were baited with pheromone lures, mechanically wounded, or left undisturbed. After 2 mo, logs were evaluated for insect feeding and the presence of S. sapinea along beetle galleries. Fresh logs were also inoculated in the greenhouse with adult I. pini that were either artificially infested or uninfested with S. sapinea spores to determine vectoring potential. Phoresy rates for individual collections ranged from 0 to 4.1%; average rates were 1.5 and 2.0% for 2004 and 2005, respectively. Isolation frequencies of S. sapinea from baited (15 ± 5%) and unbaited logs (3 ± 1%) differed significantly (P = 0.009). I. pini was also capable of transmitting the pathogen under controlled conditions. Based on phoresy rates, association, and artificial inoculation studies, we conclude that I. pini is able to transmit S. sapinea to Austrian pine stems.

Differential Response in Foliar Chemistry of Three Ash Species to Emerald Ash Borer Adult Feeding

The emerald ash borer (EAB; Agrilus planipennis Fairmaire; Coleoptera: Buprestidae), is an exotic wood-boring beetle that has been threatening North American ash (Fraxinus spp.) resources since its discovery in Michigan and Ontario in 2002. In this study, we investigated the phytochemical responses of the three most common North American ash species (black, green, and white ash) in northeastern USA to EAB adult feeding. Black ash was the least responsive to EAB adult feeding in terms of the induction of volatile compounds, and levels of only two (indole and benzyl cyanide) of the 11 compounds studied increased. In green ash, levels of two [(E)-β-ocimene and indole] of the 11 volatile compounds studied were elevated, while the levels of two green leaf volatiles [hexanal and (E)-2-hexenal] decreased. White ash showed the greatest response with an increase in levels of seven of the 11 compounds studied. Qualitative differences among ash species were detected. Among the phenolic compounds detected, ligustroside was the only one detected in all three species. Oleuropein aglycone and 2 unidentified compounds were found only in black ash; coumaroylquinic acid and feruloylquinic acid were detected only in green ash; and verbascoside hexoside was detected only in white ash. EAB adult feeding did not elicit or decrease concentrations of any selected individual phenolic compounds. However, although levels of total phenolics from black and green ash foliage were not affected by EAB adult feeding, they decreased significantly in white ash. EAB adult feeding elevated chymotrypsin inhibitors in black ash. The possible ecological implications of these findings are discussed.

The Pseudomonas syringae pv. tomato type III effector HopM1 suppresses Arabidopsis defenses independent of suppressing salicylic acid signaling and of targeting AtMIN7.

Pseudomonas syringae pv tomato strain DC3000 (Pto) delivers several effector proteins promoting virulence, including HopM1, into plant cells via type III secretion. HopM1 contributes to full virulence of Pto by inducing degradation of Arabidopsis proteins, including AtMIN7, an ADP ribosylation factor-guanine nucleotide exchange factor. Pseudomonas syringae pv phaseolicola strain NPS3121 (Pph) lacks a functional HopM1 and elicits robust defenses in Arabidopsis thaliana, including accumulation of pathogenesis related 1 (PR-1) protein and deposition of callose-containing cell wall fortifications. We have examined the effects of heterologously expressed HopM1Pto on Pph-induced defenses. HopM1 suppresses Pph-induced PR-1 expression, a widely used marker for salicylic acid (SA) signaling and systemic acquired resistance. Surprisingly, HopM1 reduces PR-1 expression without affecting SA accumulation and also suppresses the low levels of PR-1 expression apparent in SA-signaling deficient plants. Further, HopM1 enhances the growth of Pto in SA-signaling deficient plants. AtMIN7 contributes to Pph-induced PR-1 expression. However, HopM1 fails to degrade AtMIN7 during Pph infection and suppresses Pph-induced PR-1 expression and callose deposition in wild-type and atmin7 plants. We also show that the HopM1-mediated suppression of PR-1 expression is not observed in plants lacking the TGA transcription factor, TGA3. Our data indicate that HopM1 promotes bacterial virulence independent of suppressing SA-signaling and links TGA3, AtMIN7, and other HopM1 targets to pathways distinct from the canonical SA-signaling pathway contributing to PR-1 expression and callose deposition. Thus, efforts to understand this key effector must consider multiple targets and unexpected outputs of its action.

Decreased emergence of emerald ash borer from ash treated with methyl jasmonate is associated with induction of general defense traits and the toxic phenolic compound verbascoside

The emerald ash borer (EAB; Agrilus planipennis Fairmaire) is causing widespread mortality of ash (Fraxinus spp.) in North America. To date, no mechanisms of host resistance have been identified against this pest. Methyl jasmonate was applied to susceptible North American and resistant Asian ash species to determine if it can elicit induced responses in bark that enhance resistance to EAB. In particular, phenolic compounds, lignin, and defense-related proteins were quantified, and compounds associated with resistance were subsequently tested directly against EAB larvae in bioassays with artificial diet. MeJA application decreased adult emergence in susceptible ash species, comparable to levels achieved by insecticide application. Concentration of the phenolic compound verbascoside sharply increased after MeJA application to green and white ash. When incorporated in an artificial diet, verbascoside decreased survival and growth of EAB neonates in a dose-dependent fashion. Lignin and trypsin inhibitors were also induced by MeJA, and analogs of both compounds reduced growth of EAB larvae in artificial diets. We conclude that the application of MeJA prior to EAB attack has the ability to enhance resistance of susceptible ash trees by inducing endogenous plant defenses, and report evidence that induction of verbascoside is a mechanism of resistance to EAB.

Nutritional attributes of ash (Fraxinus spp.) outer bark and phloem and their relationships to resistance against the emerald ash borer

The emerald ash borer (Agrilus planipennis Fairmaire, EAB) is an alien, invasive wood-boring insect that is responsible for killing millions of ash trees since its discovery in North America in 2002. All North American ash species (Fraxinus spp.) that EAB has encountered have shown various degrees of susceptibility, while Manchurian ash (Fraxinus mandshurica Ruprecht), which shares a co-evolutionary history with this insect, is resistant. Recent studies have looked into constitutive resistance mechanisms in Manchurian ash, concentrating on the secondary phloem, which is the feeding substrate for the insect. In addition to specialized metabolism and defense-related components, primary metabolites and nutritional summaries can also be important to understand the feeding behavior of insect herbivores. Here, we have compared the nutritional characteristics (water content, total protein, free amino acids, total soluble sugars and starch, percent carbon and nitrogen, and macro- and micronutrients) of outer bark and phloem from black, green, white and Manchurian ash to determine their relevance to resistance or susceptibility to EAB. Water content and concentrations of Al, Ba, Cu, Fe, K, Li, tryptophan and an unknown compound were found to separate black and Manchurian ash from green and white ash in a principal component analysis (PCA), confirming their phylogenetic placements into two distinct clades. The traits that distinguished Manchurian ash from black ash in the PCA were water content and concentrations of total soluble sugars, histidine, lysine, methionine, ornithine, proline, sarcosine, tyramine, tyrosol, Al, Fe, K, Na, V and an unknown compound. However, only proline, tyramine and tyrosol were significantly different, and higher, in Manchurian ash than in black ash.