Steven A. Whitham

The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor

The products of plant disease resistance genes are postulated to recognize invading pathogens and rapidly trigger host defense responses. Here we describe isolation of the resistance gene N of tobacco that mediates resistance to the viral pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator transposon. A genomic DNA fragment containing the N gene conferred TMV resistance to TMV susceptible tobacco. Sequence analysis of the N gene shows that it encodes a protein of 131.4 kDa with an amino-terminal domain similar to that of the cytoplasmic domain of the Drosophila Toll protein and the interleukin-1 receptor (IL-1R) in mammals, a nucleotide-binding site (NBS), and 14 [corrected] imperfect leucine-rich repeats (LRR). The sequence similarity of N, Toll, and IL-1R suggests that N mediates rapid gene induction and TMV resistance through a Toll-IL-1-like pathway.

Global Impact: Elucidating Plant Responses to Viral Infection

Viruses induce a variety of responses in host cells that are mediated by perturbation of different signaling pathways. Advances in our understanding of the functions of viral proteins, plant biology in general, as well as technologies for profiling gene expression have converged in recent years to provide new insight into the events occurring inside susceptible and resistant host cells in response to virus infection. These effects range from nonspecific changes in gene expression due to the general accumulation of viral proteins to those responses that are initiated by the specific interactions between virus and host proteins. Here, we discuss a variety of expression profiling methods and approaches that have been used to study the effects of viruses on host transcriptomes. These studies have identified distinct sets of genes that have altered expression profiles in response to viruses, including stress- and defense-related genes. The activities of viral RNA silencing suppressors and interference with hormone signaling or biogenesis also influence plant gene expression and lead to developmental abnormalities.

A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

Spatial Analysis of Arabidopsis thaliana Gene Expression in Response to Turnip mosaic virus Infection

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R1 and R2 generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.

Gene silencing and DNA methylation processes

Virus-infected leaf tissues comprise a heterogeneous mixture of cells at different stages of infection. The spatial and temporal relationships between sites of virus accumulation and the accompanying host responses, such as altered host gene expression, are not well defined. To address this issue, we utilized Turnip mosaic virus (TuMV) tagged with the green fluorescent protein to guide the dissection of infection foci into four distinct zones. The abundance of Arabidopsis thaliana mRNA transcripts in each of the four zones then was assayed using the Arabidopsis ATH1 GeneChip oligonucleotide microarray (Affymetrix). mRNA transcripts with significantly altered expression profiles were determined across gradients of virus accumulation spanning groups of cells in and around foci at different stages of infection. The extent to which TuMV-responsive genes were up- or downregulated primarily correlated with the amount of virus accumulation regardless of gene function. The spatial analysis also allowed new suites of coordinately regulated genes to be identified that are associated with chloroplast functions (decreased), sulfate assimilation (decreased), cell wall extensibility (decreased), and protein synthesis and turnover (induced). The functions of these downregulated genes are consistent with viral symptoms, such as chlorosis and stunted growth, providing new insight into mechanisms of pathogenesis.

Salicylic Acid-Dependent Expression of Host Genes in Compatible Arabidopsis-Virus Interactions

Epigenetic gene silencing results from the inhibition of transcription or from posttranscriptional RNA degradation. DNA methylation is one of the most central and frequently discussed elements of gene silencing in both plants and mammals. Because DNA methylation has not been detected in yeast, Drosophila or Caenorhabditis elegans, the standard genetic workhorses, plants are important models for revealing the role of DNA methylation in the epigenetic regulation of genes in vivo.

Distinct Biphasic mRNA Changes in Response to Asian Soybean Rust Infection

Plant viruses elicit the expression of common sets of genes in susceptible hosts. Studies in Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) indicate that at least one-third of the genes induced in common by viruses have been previously associated with plant defense and stress responses. The genetic and molecular requirements for the induction of these stress and defense-related genes during compatible host-virus interactions were investigated with a panel of Arabidopsis mutant and transgenic plants defective in one or more defense signaling pathways. pad4, eds5, NahG, npr1, jar1, ein2, sid2, eds1, and wild-type Columbia-0 and Wassilewskija-2 plants were infected with two different viruses, cucumber mosaic virus and oilseed rape mosaic virus. Gene expression was assayed by a high-throughput fiber-optic bead array consisting of 388 genes and by RNA gel blots. These analyses demonstrated that, in compatible host-virus interactions, the expression of the majority of defense-related genes is induced by a salicylic acid-dependent, NPR1-independent signaling pathway with a few notable exceptions that did require NPR1. Interestingly, none of the mutant or transgenic plants showed enhanced susceptibility to either cucumber mosaic virus or oilseed rape mosaic virus based on both symptoms and virus accumulation. This observation is in contrast to the enhanced disease susceptibility phenotypes that these mutations or transgenes confer to some bacterial and fungal pathogens. These experimental results suggest that expression of many defense-related genes in compatible host plants might share components of signaling pathways involved in incompatible host-pathogen interactions, but their increased expression has no negative effect on viral infection.

The Unfolded Protein Response is Triggered by a Plant Viral Movement Protein

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.

Identification and Analyses of Candidate Genes for Rpp4-Mediated Resistance to Asian Soybean Rust in Soybean

Infection with Potato virus X (PVX) in Nicotiana benthamiana plants leads to increased transcript levels of several stress-related host genes, including basic-region leucine zipper 60 (bZIP60), SKP1, ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT), and calmodulin (CAM). bZIP60 is a key transcription factor that responds to endoplasmic reticulum (ER) stress and induces the expression of ER-resident chaperones (BiP, PDI, CRT, and CAM). SKP1 is a component of SCF (for SKP1-Cullin-F box protein) ubiquitin ligase complexes that target proteins for proteasomal degradation. Expression of PVX TGBp3 from a heterologous vector induces the same set of genes in N. benthamiana and Arabidopsis (Arabidopsis thaliana) leaves. Virus-induced gene silencing was employed to knock down the expression of bZIP60 and SKP1, and the number of infection foci on inoculated leaves was reduced and systemic PVX accumulation was altered. Silencing bZIP60 led to the suppression of BiP and SKP1 transcript levels, suggesting that bZIP60 might be an upstream signal transducer. Overexpression of TGBp3 led to localized necrosis, but coexpression of TGBp3 with BiP abrogated necrosis, demonstrating that the unfolded protein response alleviates ER stress-related cell death. Steady-state levels of PVX replicase and TGBp2 (which reside in the ER) proteins were unaltered by the presence of TGBp3, suggesting that TGBp3 does not contribute to their turnover. Taken together, PVX TGBp3-induced ER stress leads to up-regulation of bZIP60 and unfolded protein response-related gene expression, which may be important to regulate cellular cytotoxicity that could otherwise lead to cell death if viral proteins reach high levels in the ER.