Tobacco mosaic virus (TMV) • RNA virus discovered by Beijerinck over 100 years ago, as a contagium vivum fluidum that was filterable and could move through agar media. (Bacteria were not filterable and remained fixed in agar). • CTMV causes mosaic disease in tobacco and other plants. • Widely used in host-pathogen research.
Why TMV? • Mosaic disease of tobacco was detrimental to the tobacco industry in the early 1900’s. • Mayer in 1886, in the Netherlands, was the first to artificially transmit a plant disease (TMV), the causal agent of which could not be seen or cultured. • Around the same time, Pasteur was having a similar problem with rabies!
Model Systems • Models become the driving force for independent steps of discovery, intervention, and development. • TMV rapidly accumulates to high titres in infected plants. • TMV is not transmitted by insects, fungi, or nematodes, but is by rub-inoculation. • TMV symptoms are easy to identify and can infect a wide range of plants.
TMV in the laboratory • Easy, cheap, and reliable to produce. • Omega leader sequence has been widely used to enhance translation of foreign genes in transgenic plants. • cDNA copies of TMV promoters have been used in gene vectors for expression of foreign genes in rub-inoculated plants.
Pathogenic Plant Bacteria • Pseudomonas syringae pv. tomato DC3000 • Bacterial speck disease caused on tomato and mustard plants. • >30 Type III Secretion System effectors.
Arabidopsis thaliana • Just as animal biologists and medical researchers have used model organisms such as Drosophila and the nematode, so plant biologists have relied upon the mustard Arabidopsis thaliana to help them understand the genetics, physiology, development and structure of plants in general. • Although the efforts of Arabidopsis sequencers have perhaps been less heralded than those of their colleagues working on animal genomes, the results they have yielded are no less scientifically interesting. Arabidopsis may be a simple plant (its detractors call it a weed!) with only 50 identified cells types and no complex nervous system or behavioural response, but its genome is surprisingly complex. Chromosomes 2 and 4 of Arabidopsis were published in Nature at the end of 1999, now followed by chromosomes 1, 3 and 5. http://www.nature.com/genomics/papers/a_thaliana.html
Plant Defenses • Basal defenses: • Cell wall reinforcements • Expression of defense-associated proteins • Activated when LPS or flagellin detected. • Intracellular Resistance (R) Proteins: • Detect Type III effectors in plant cells • Hypersensitive Response (HR) induces Programmed Cell Death (PCD).
Pseudomonas DC3000 • Full genome has been sequenced. • DC3000 elicits HR immunity (via apoptosis) in plants carrying Pto R proteins (serine/threonine kinase). • Plant Pto interacts with bacterial DC3000 AvrPto and AvrPtoB effectors as well as Prf for HR response and plant immunity.
Plants without Pto protein • Without Pto, the effectors AvrPto and AvrPtoB promote bacterial growth! • AvrPto suppresses cell wall-based defenses. • AvrPtoB has Cell Death Suppressor (CDS) activity to prevent HR-based PCD (apoptosis).
AvrPtoB • Belongs to VirPphA gene family. • VirPpha is a virulence factor located on a large Pph plasmid. • VirPpha and AvrPtoB have 51% sequence identity, so they are most likely related but not homologous proteins. • AvrPtoB is bigger than AvrPto but has a similar interaction with Pto.
Cell Death Suppression • AvrPtoB CDS was discovered with Nicotiana, because of its robust cell death response. • AvrPto with Pto results in PCD, but AvrPtoB with Pto in Nicotiana did not result in Programmed Cell Death! • Mutant and pro-apoptotic experiments with AvrPtoB supported CDS activity. • Hidden R gene discovered: • Rsb = Resistance Suppressed by the avrPtoB C-terminus involved in CDS activities.
DC3000 Pathogenesis • Wild type tomato plants with AvrPtoB and without functional pto gene = CDS. • Mutant AvrPtoB led to PCD and immunity. • Introducing AvrPtoB in trans restored virulence of DC3000 and CDS!
Rsb gene • AvrPtoB must be recognized by at least two resistance genes. Rsb is Pto related: • Like Pto, Rsb is Prf dependent for CDS. • Gene silencing experiments of Pto family members eliminates Rsb resistance in tomatoes and PCD in Nicotiana plants.
Other DC3000 Effectors • HoPtoD2, HopPtoE, HopPtoF, HopPtoN, AvrPphE, AvrPpiB1… • Cell Death Suppression activity is not specific and can inhibit various R proteins. • Most effectors were discovered based on eliciting the HR immune response, but they also have the potential for CDS activity.
Trump Model • Not the Donald, thank goodness! • The host-pathogen interaction may involve an R protein as well as a Trump factor that can suppress the CDS activity. • This model accounts for the ‘hidden resistance genes’ and complex plant-bacteria interactions.
Trump Model Version 1 • The CDS effector is dominantly expressed if R protein but no T factor is present.
Trump Model Version 2 • CDS effector is suppressed if both R protein and T factor are present.
Trump Model Version 3 • CDS effector is dominantly expressed if T factor but no R protein present.
Other Virulence Activities • AvrPtoB • DC3000 mutants without CDS activity cause a 10-fold growth decrease in tomato plants. • Plants with prf mutations cannot mount Pto- or Rsb-mediated immunity. • AvrPtoB can activate ethlyene production in tomato plants, increasing cell death.
Why would bacteria evolve to both enhance and suppress cell death? • Perhaps early in infection it is advantageous to suppress PCD to escape the immune system and establish the infection. • Later in the infection it may be advantageous to enhance PCD to gain access to new cells and nutrients.
Phytophthora ramorum • First observed causing bleeding stem cankers and mortality of tanoak and coast live oak in coastal CA in the 1990’s, it has now been identified in North America and Europe. • Mortality associated with Fagus genus but foliar and twig infections of other species are important for pathogen spread.
Sudden Oak Death Research • Individual tree level • Forest level • Regional level
Forest Succession • Mosaic landscapes provide a variety of niches and microclimates. • Successional stages follow disturbances such as fire or logging. • Tanoak sprouts prolifically following fire, and is later replaced by Douglas-fir or coast redwood trees.