Joel J. Milner

Cauliflower mosaic virus, a Compatible Pathogen of Arabidopsis, Engages Three Distinct Defense-Signaling Pathways and Activates Rapid Systemic Generation of Reactive Oxygen Species

We analyzed expression of marker genes for three defense pathways during infection by Cauliflower mosaic virus (CaMV), a compatible pathogen of Arabidopsis (Arabidopsis thaliana), using luciferase reporter transgenes and directly by measuring transcript abundance. Expression of PR-1, a marker for salicylic acid signaling, was very low until 8 d postinoculation and then rose sharply, coinciding with the rise in virus levels. In contrast, as early as 2 h postinoculation, transcriptional up-regulation of GST1—a marker for reactive oxygen species—and PDF1.2—a marker for jasmonic acid/ethylene defense signaling—was detectable in the virus-inoculated leaf and systemically. In parallel with the activation of GST1, H2O2 accumulated locally and systemically in virus- but not mock-inoculated plants. However, in plants inoculated with infectious CaMV DNA rather than virus particles, the onset of systemic luciferase activity was delayed by 24 to 48 h, suggesting that virion structural proteins act as the elicitor. This phenomenon, which we term the rapid systemic response, preceded virus movement from the inoculated leaf; therefore, the systemic signal is not viral. Systemic, but not local, H2O2 accumulation was abolished in rbohDF double mutants and in etr1-1 and ein2-1 mutants, implicating NADPH oxidase and ethylene signaling in the generation and transduction of the response. Ethylene, but not rbohDF mutants, also showed reduced susceptibility to CaMV, whereas in NahG transgenics, virus levels were similar to wild type. These findings implicate reactive oxygen species and ethylene in signaling in response to CaMV infection, but suggest that salicylic acid does not play an effective role.

Manipulation and trapping of sub-micron bioparticles using dielectrophoresis

A non-uniform alternating electric field induces motion in polarisable particles called dielectrophoresis. The effect is governed by the relative magnitudes of the dielectric properties of the medium and the particles. The technology has been used to manipulate particles for biotechnological applications, including purification, fractionation and concentration of cells and microorganisms. However, the lower size limit for the dielectrophoretic manipulation of particles was believed to be about 1 micron, but recent work has proved otherwise. The dielectrophoretic movement and properties of latex beads and a simple rod-shaped virus, tobacco mosaic virus (TMV), have been measured using microfabricated electrode structures. Measurements have been made over a range of suspending medium conductivities, applied frequencies and electric field strengths. It is shown that under appropriate conditions both latex beads and tobacco mosaic virus particles can be selectively attracted to regions of high electric field strength located at the tips of microfabricated electrode structures. The ability to selectively trap and separate bio-particles has many potential applications in the area of biotechnology.

Timing is everything: regulatory overlap in plant cell death.

Plant development and defence are intimately connected to programmed cell death (PCD). PCD can occur after environmental cues such as pathogen infection, mechanical damage or abiotic stress. However, PCD also constitutes an essential feature of various aspects of growth and development. Despite the differences in stimuli, the subsequent steps leading to programmed cellular death show considerable commonality, reflecting the essential and overlapping roles of individual regulatory components in these processes. These components can function as positive or negative regulators and can have contrasting functions depending on the form of cell death.

Transgenic Arabidopsis lines expressing gene VI from cauliflower mosaic virus variants exhibit a range of symptom-like phenotypes and accumulate inclusion bodies

Gene VI of cauliflower mosaic virus (CaMV) is an important determinant of symptom expression during infection. We have constructed a series of transgenic Arabidopsis lines that express gene VI protein (P6) from two CaMV isolates (Bari-1 and Cabb B-JI) that cause mild and severe symptoms, respectively, in Arabidopsis, and from a recombinant virus (Baji-31) with a hybrid gene VI that causes very severe symptoms. From 41 transgenic lines analyzed, 17 showed symptom-like phenotypes that ranged from mild vein chlorosis to severe chlorosis and stunting. P6 levels in transgenic lines varied from undetectable in the lowest expressors to levels greater than those in CaMV-infected plants. There was a strong correlation between phenotype severity and the level of P6, and with the gene VI origin in the order, Baji-31 > B-JI > Bari-1. This was similar to symptom severity in Arabidopsis infected with the respective CaMV variant. We also found that transgenic P6 accumulated in inclusion bodies that were similar to those found in infected plants but lacking virions. We conclude that expression of P6, in the absence of virus replication, elicits a subset of the host symptom responses normally observed during infection and that the level, sequence, and possibly the form of P6 are important in potentiating the process.

Cauliflower mosaic virus Protein P6 Inhibits Signaling Responses to Salicylic Acid and Regulates Innate Immunity

Cauliflower mosaic virus (CaMV) encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling) and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst). Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity) suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls) but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants. These results demonstrate that P6 is a new type of pathogenicity effector protein that enhances susceptibility to biotrophic pathogens by suppressing SA- but enhancing JA-signaling responses.

Components of Arabidopsis Defense- and Ethylene-Signaling Pathways Regulate Susceptibility to Cauliflower mosaic virus by Restricting Long-Distance Movement

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.

Altered Patterns of Gene Expression in Arabidopsis Elicited by Cauliflower Mosaic Virus (CaMV) Infection and by a CaMV Gene VI Transgene

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.

Ferredoxin Containing Bacteriocins Suggest a Novel Mechanism of Iron Uptake in Pectobacterium spp

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium Pectobacterium carotovorum carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of Pectobacterium carotovorum and Pectobacterium atrosepticum with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that Pectobacterium spp. carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of Pectobacterium carotovorum and atrosepticum that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells.

Dielectric spectroscopy of Tobacco Mosaic Virus

The dielectric properties of the Tobacco Mosaic Virus (TMV) have been measured using time domain dielectric spectroscopy (TDDS) in the temperature range from 1 to 40 degrees C. A single dielectric dispersion is observed in the MHz range. The activation energy of the process is found to be in the range 1-2 kcal/mol. The experimental data could not be completely accounted for by current theoretical models, but evidence indicates that the dielectric loss arises from polarisation of charge on and around the virus.

Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis.

Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35Stms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures.