PLP 535 Lecture

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PLP 535 Lecture

• Lee A Hadwiger

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Lecture535jan08

• Defense responses
• The next two lecture periods involve signaling
  and the biochemistry and genetics of defense
  responses, primarily the nonhost resistance
  response.
• The student will be responsible for two review
  articles and one journal article for presentation
  by the student assigned to this topic. They are
  as follows
• Ellis J. Insights in to nonhost disease
  resistance Can they assist disease control in
  agriculture? 2006. The Plant Cell 18523-528.
• Hadwiger, L. A. 2008. Pea/Fusarium solani
  interactions contributions of a system towards
  understanding disease resistance. Phytopathology
  (IN PRESS).
• Student presentation article
• Stein, M. et al. 2006 Arabidopsis PEN3/PDR8, an
  ATP binding cassette transporter, contributes to
  nonhost resistance to inappropriate pathogens
  that enter by direct penetration. Plant Cell
  128731-746.

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• Text of the two lecture periods
• During evolution, a pathogen is a pathogen
  because it has found a niche on a given plant
  where it can grow and multiply. This niche can
  be destroyed or altered by a change in the host
  plant and in modern agriculture it is often
  messed up and most often by a breeding program
  that has introduced a new single dominant gene.
  The resistance expressed by this gene is
  dependent on a signal. This signal can be
  encoded by an avirulence gene, a loss of this
  gene in the pathogen again allows the pathogen to
  become virulent. In most instances this loss
  represents a loss of function. It is this loss
  of function that allows the pathogen to become
  virulent. The cloning of host R genes and
  pathogen avirulence (Avr) genes has been
  accompanied by a simplistic view of host-parasite
  interactions. Physiologically speaking, the
  appropriate match of the gene for gene
  interaction starts a process that is the straw
  that breaks the camels back. That is it is
  the apex of signaling events that occur following
  the contact between host and fungus, even though
  the match-up is the ultimate decision maker that
  determines that an incompatible reaction will
  occur.

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Quadratic check

• Pathogen genes
• Host genes PP or Pp pp
• RR or Rr Resistance Susceptible
• rr Susceptible
  Susceptible

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Terminology slide, spore, germ tube,
appressorium, haustorium
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Nonhost resistance(Inappropriate host)

• You have previously been introduced to host
  resistance controlled by R genes against races of
  true pathogens (appropriate pathogens) on a given
  host. If the true pathogen challenges another
  plant, (what is now considered an inappropriate
  pathogen on a nonhost) the defense response is
  intense and in almost all cases is a plant
  defense that does not break down under field
  conditions.

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Quadratic check in non-host resistance model

• Fusarium Pathogens
• Host F. solani f. sp. phas. F.s. f. sp pisi
• Pea Resistance react. Suscept.
  Bean Suscept. react. Resist
  

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NON-HOST RESISTANCE

• Nonhost resistance responses are triggered by
  many more decision-making signals and most
  likely involving many more genes from both host
  and pathogen. There has been no demonstration of
  one gene being the key to the success or demise
  of the pathogen, however, some mutants come
  close. In Arabidopsis there is a mutant termed
  npr1 (no PR proteins) that comes close. This
  mutant hits a major function in the host
  metabolism that prevents multiple genes (PR
  genes) from being expressed

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• .
• It has been established long ago, that protein
  synthesis in general is required for disease
  resistance in both nonhost and race specific
  resistance. The best demonstration coming from
  my lab. of how to break resistance was by the use
  of heat shock. Heat shock of both plants and
  animals is accompanied by a shift of protein
  synthesis from normal metabolism to activation of
  heat shock genes. When pea tissue is heat
  shocked (only one hour of 35 C temperature is
  required to this) it produces the mRNA for heat
  shock protein for about 9 hour with the
  exclusion of PR and other proteins. During this
  time the tissue is totally susceptible to
  inappropriate pathogens and unable to activate
  its PR genes to get PR proteins. After 9 hours
  the tissue returns to normal and its ability to
  resist inappropriate pathogens returns along with
  its ability to produce PR proteins.

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RNA and Protein synthesis-for PR protein
production

• RNA synthesis inhibitors
• Cordycepin- blocks mRNA transport
• Alpha- amanitin - blocks RNA polymerase
• Protein synthesis inhibitors
• Cycloheximide - blocks protein synthesis at the
  ribosomal level.

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Inhibitors

• We have also demonstrated the ability of
  specific inhibitors of enzymes controlling major
  functions in the cell to completely block disease
  resistance. There are several phosphatase
  inhibitors that work apparently by blocking major
  cell functions (phosphorylations) required for
  resistance. In early work we demonstrated that
  simply blocking total gene transcription
  (including PR genes) and translation (including
  PR proteins) it readily blocked disease
  resistance.

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Toxins metabolic blocking and membrane damaging

• There are some plant pathogens that bring along
  mechanisms to block aspects of the host response
  and are called toxins - and toxins come in a
  variety of chemical forms. Thus there are many
  different targets in the host plant cell for
  disruptions that can block the disease
  resistance response.

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Quadratic check- host specific toxins

• oat pathogen toxin corn path. Toxin
• Host
• Oat Susceptible react. Resist. Reaction
• Corn Resist. Reaction Suscept. React.
• Critical research -Scheffer E. E. Nelson

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Time course of events in non-host resistance in
peas

• 20 minutes - alteration of nuclear density
• 1 hour - DNA alteration
• 2 hours - detection of PR gene transcription
• 2-3 hours- phosphorylation HMG-I
• 5 hours - suppression of fungal growth
• 6 hours - pisatin synthesis
• 10 hours - increase in chitinase, glucanase
• 18 hours - H.S., Cell death

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BIOCHEMISTRY OF RESISTANCE
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Chitosan and chitin are examples of
PAMPs or MAMPs
. Other cell
wall fragments are considered, Microb/pathoge
n
-
Chitin chitosan
Associated molec. patterns
.
Previously known as elicitors
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Pea DRR206 is on this pathway
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PR genes for PAL, CHS, and others to Pisatin on
this pathway
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Mutational genetics of nonhost resistance

• The Arabidopsis researchers have used the Ti
  plasmid to mutate about every gene in this plant
  and some looked for a break-down of nonhost
  resistance.
• Three mutant lines have been heralded as the
  major players in nonhost resistance. The genes
  are PEN1.PEN2 and PEN3.

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Mutational genetic technology

• The scientific approach used by many is to
  screen for a functional loss. The gene in the
  vicinity of the hit is cloned and sequenced.
  Then you go to the GenBank and see if the
  sequence of your mutated gene has a match. If it
  does, its function is called putative. You are
  then free to insert that function into a disease
  resistance model.
• With modern gene manipulations you can also
  locate and sequence the genes promoter region.
  This will enable one to find sequences that are
  recognized by certain transcription factors.
  The promoter region can be further analyzed by
  making constructs that can contain Various
  segments of the promoter, various combinations of
  the open reading frame (ORF), attachments
  containing one of many combinations of reporter
  genes, such a green fluorescent protein, GUS etc.
  Also the open reading frame can be extended with
  a sequence that will produce a peptide that can
  be recognized by an anti-sera that is
  commercially produced.

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Immunological technology

• An anti-sera can be developed that will recognize
  the product of the open reading frame. Having
  the sequence of the ORF one can chose a predicted
  amino acid segment deemed to possess antigenic
  determinant properties. This segment can be
  synthesized and injected into a rabbit or other
  suitable animal to generate anti-sera specific to
  the coded protein. The entire ORF or parts of it
  can be inserted into an expression vector system
  and allow the bacterium to produce abundant gene
  product. The product can be purified and then
  injected into the rabbit to develop anti-sera.
• With these tools there an abundance of
  techniques that will help understand the function
  of the gene detected. One of the first/best
  approaches is to over express the gene in the
  plant and determine if the greater abundance
  affects disease resistance or other functions.
  With the use of the flag peptide-antisera, the
  gene fluorescent protein (GFP) marker or the
  total gene specific antisera it is possible to
  follow the cytological location of the protein in
  a living cell.

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PEN gene putative functions

• PEN1 has homology with Syntaxin
• PEN2 - has homology with glycosyl hydrolase, one
  of a large family.
• PEN 3 encodes a putative ATP binding cassette
  (ABC) transporter PDR8. It localizes in the
  plasma membrane and may mediate targeted exports
  of toxins to the penetration site. Details of
  these mutants will be covered in the student
  lecture.
• Bottom line each mutant enables the pathogen to
  progress some, but none enables total
  susceptibility

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Genetic engineering

• Systems
• Particle gun
• Agrobacterium tumefaciens T-I plasmid
• Success See Punja review article

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Engineering approaches

• Use TI plasmid to insert the following
  constructs
• 35S promoter- PR gene
• 35S promoter R gene
• 35S promoter intermediate enzyme to phytoalexin
• 35S promoter any other vital step
• Inducible promoter for any of the above
• Promoter for anti-sense or siRNA sequences
• Promoter for syntheses of or blocking receptor
  proteins
• Unconventional constructs.
• Promoter from DRR206 driving F. solani f. sp
  phaseoli DNase gene

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Thought evolution of the
biochemistry of disease resistance

• Totally chemistry
• When J. C. Walker discovered the purple skins of
  an onion protected it from pathogens. Most
  everyone did chemical analyses of plants looking
  for resistance. Unfortunately this type of
  disease resistance was unique to onions and to
  one group of pathogens.

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Nutrition hypothesis

• Pathologists proposed that the reason that only
  the appropriate pathogen grew on the appropriate
  host was that the host had the right nutrients
  (biochemists were involved at that time in
  analyzing the metabolism of amino acids, sugars
  etc.) True, all pathogens need nutrition but
  most fungi could use very basic N,P,K, minor
  elements, carbon source to grow. However most
  non-obligate pathogens grew better with amino
  acid/sugar supplements but these could be found
  in and around most plants at least in low amounts
  and around germinating seeds in larger amounts.
  It was difficult to establish that nutrition was
  the basis for the appropriate pathogen growing on
  the appropriate host.

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Phytoalexin hypothesis

• An extension of the phenolics thinking. Plants
  ward off the inappropriate pathogens by producing
  phytoalexins (small compounds that possess
  antifungal action in vitro). Again, the
  hypothesis was true to a point. Most defense
  responses are accompanied by accumulations of
  phenolics. Often the accumulation is
  proportional to the resistance expressed.
  However there are some exceptions
• 1. Some plants with abundant phytoalexins
  accumulations are still susceptible.
• 2. In pea tissue the inappropriate pathogen
  growth is suppressed before there is detectable
  accumulations of phytoalexins.
• A major benefit of phytoalexins research was the
  discovery that the accumulation of phytoalexins
  was due to the induction of genes that encode
  enzymes that are in secondary pathways.

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Induction hypothesis

• There was skepticism when Martin Schwochau and I
  devised the induction hypothesis to explain how
  the match ups in gene-for-gene interaction
  generated resistance that was epistatic to all
  other match-ups, even though many other genes
  were present. A man named Art Browning had
  popularized a lock and key model that was widely
  taught, was very complicated and wrong.

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Specific inhibitors reduce skepticism

• Much of the skepticism of the induction
  hypothesis was erased when resistance was shown
  to be blocked at three levels with specific
  inhibitors
• Alpha amanitin blocked DNA-dependent RNA
  polymerase II.
• Cordycepin blocked the transfer of mRNA from
  the nucleus.
• Cycloheximide blocked the translation of mRNA
  into protein at the ribosomal level.
• These inhibitors also worked nicely to indicate
  when each event was occurring . That is, you
  could block transcription and stop the
  development of the resistance response if it was
  applied within the first hour or so.
  Applications after 6 hours were no longer
  effective. Cordycepin applied after 3- 6 hours
  was ineffective and cycloheximide could still be
  effective at 4 hours in blocking resistance.

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PR proteins, what do they do?

• PR PROTEINS
• What do PR proteins do?
• 1. Many are associated with secondary plant
  metabolism primarily with the synthesis of
  phenolics such as flavonoids, isoflavonoids,
  salicylic acid, catechol, tannins, lignin and
  suberin.
• Other compounds are synthesized via other
  pathways such as terpenes and alkaloids.
• 2. Hydrolytic enzymes proteases, RNases,
  chitinases, ß-glucanases, lipases etc. The
  fragmentation products ot these enzymes can also
  signal other responses, e.g. chitin and chitosan
  oligomers.
• 3. High Cysteine proteins (peptides).
  Defensins and other anti-fungal proteins.
• 4. Enzymes involved in cell wall
  contruction/degradation Cellulose synthase,
  callose synthesis, etc.
• 5. Many house keeping genes. These are picked
  up in micro array analyses and can have a variety
  of functions related to ionization, super oxide
  development transport etc.

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Observations distinquishing the pea system from
Arabidopsis

• 1. The pea endocarp tissue that does not have a
  cuticle barrier, can rapidly distinquish between
  the true (appropriate) pea pathogen, Fusarium
  solani f. sp. pisi and the inappropriate pathogen
  (Bean pathogen) Fusarium solani f. sp. phaseoli
  (nonhost resistance).
• The bean pathogen growth is stopped at 6 hours
  post inoculation. The signaling between pathogen
  and host is rapid since the newly exposed pea
  endocarp tissue has no cuticle layer.
• Note Surprisingly, plant scientists were
  reluctant until the last decade or so to believe
  that anything but small molecular compounds could
  penetrate the plant cell wall and enter the
  cytoplasm. Now they concede that compounds in
  the vicinity of 30 kD can enter without a carrier
  system.
• 2. Other potential barriers in peas such as
  super oxides, nitric oxide, salicylic acid (SA)
  and jasmonic acid (JA) induced responses, the
  hypersensitive response (HR), programmed cell
  death (apoptosis) and phytoalexins accumulation
  are probably not initially involved in
  resistance, since they do not compromise a part
  of the pea cells response when subjected to
  an inhibitor study. Also many of these processes
  do not appear within the first 6 hours after
  inoculation, a time when the inappropriate
  pathogen is totally stopped.

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A well studied phenolic- pisatin

• Dr. Hans VanEtten devoted his years to the study
  of pisatin and is currently looking at the
  biosynthetic route of pisatin biosynthesis. His
  major premise was, one reason that a pathogen
  becomes an appropriate pathogen of peas is that
  it is capable of degrading pisatin the single
  phytoalexin found in peas. I will refer you to
  the assigned mini review that was reviewed by Dr.
  VanEtten for the complete story.

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Pisatin some bottom lines

• Some bottom lines
• 1. Pisatin delays germination of F. solani f.
  sp. phaseoli/pisi for 24 hours.
• 2. Pisatin accumulation is more rapid in lines
  possessing R genes.
• 3. Pathogenic isolates of F. solani f. sp. pisi
  capable of more rapidly degrading pisatin, appear
  to be more virulent.
• 4. Pathogens mutated to no longer produce the
  enzyme p450 (pisatin demethylase) are less
  virulent.
• 5. A complete loss of pisatin degradation
  potential in the pathogen does not completely
  reduce virulence.
• 6. The Fusarium solani f. sp. pisi pathogen has
  virulence traits that are located on auxiliary
  chromosomes, that have no relationship with
  pisatin degradation.

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What does stop fungal growth on the plant surface
?

• Much of the current literature is concentrated on
  superoxides , nitric oxide, jasmonic acid
  pathway products, salicylic acid pathway
  products, programmed cell death, callose
  accumulations, lignin accumulations, etc. which
  will be covered by other lecturers.
• It is likely that disease resistance in
  Arabidopsis differs from that in peas and other
  legumes. Along with differences in other plant
  systems, will come differences in view-points
  based on that research. My remarks reflect my
  research on peas and the limited use of pea PR
  genes transferred to other plants.

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Research at odds with arabidopsis

• 1. The pea endocarp tissue that does not have a
  cuticle barrier, can rapidly distinquish between
  the true (appropriate) pea pathogen, Fusarium
  solani f. sp. pisi and the inappropriate pathogen
  (Bean pathogen) Fusarium solani f. sp. phaseoli
  (nonhost resistance).
• The bean pathogen growth is stopped at 6 hours
  post inoculation. The signaling between pathogen
  and host is rapid since the newly exposed pea
  endocarp tissue has no cuticle layer.
• Note Surprisingly, plant scientists were
  reluctant until the last decade or so to believe
  that anything but small molecular compounds could
  penetrate the plant cell wall and enter the
  cytoplasm. Now they concede that compounds in
  the vicinity of 30 kD can enter without a carrier
  system.

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The first cloning of a fungal Avirulence gene was
accomplished by the De Wit laboratory. Avr 9
product is a 28 amino acid residue peptide with 3
disulfide bridges. When the gene coding this
peptide in bred into a tomato plant containing
the Cf-9 R-gene, the plant dies. See page 1123
of the Biochemistry and Molecular Biol. Of Plants
Text. Figure gives 3 (p. 1128) possibilities for
how the peptide activates defense. Recently the
direct contact option is no long a possibility.
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The mlo gene controlled by a recessive trait

• The mlo gene is the only plant resistance gene
  that has been selected for following the
  mutagenesis of a susceptible wild-type (Mlo)