Role of enzymes in pathogenesis

Pathogenesis Level-3 Semester-2

1. Lecture Notes
For Level-3 Semester-2
Prepared by
Prof. Dr. Md. Rashidul Islam
Department of Plant Pathology
Bangladesh Agricultural University
Mymensingh

2. The first contact of pathogens with their host plants occurs at a
plant surface. Aerial plant partsurfaces consist primarily of cuticle
and/or cellulose, whereas root cell wall surfaces consist only of
cellulose.
Cuticle consists primarily of cutin, more or less impregnated
with wax and frequently covered with a layer of wax. The lower
part of cutin is intermingled with pectin and cellulose lamellae and
lower yet there is a layer consisting predominantly of pectic
substances;below that there is a layer of cellulose.
Polysaccharides of various types are often found in cell walls.
Proteins of many different types, both structural, e.g., elastin,
which helps loosen the cell wall, and extensin, which helps add
rigidity to the cell wall, some enzymes, and some signal molecules
that help receive or transmit signals inward or outward, are normal
constituents of cell walls.
Finally, epidermal cell walls may also contain suberin and lignin.
Enzymes in Pathogenesis

3. Thepenetrationofpathogensintoparenchymatoustissuesis
facilitatedbythebreakdownoftheinternalcellwalls,which
consistofcellulose,pectins,hemicelluloses,andstructural
proteins,andofthemiddlelamella,whichconsistsprimarilyof
pectins.
Inaddition,completeplanttissuedisintegrationinvolvesthe
breakdownoflignin.
Thedegradationofeachoftheabovementionedsubstances
isbroughtaboutbytheactionofoneormoresetsofenzymes
secretedbythepathogen.

4. Cutinases
Schematicrepresentationofthestructureandcompositionofthe
cuticleandcellwalloffoliarepidermalcellsalongwiththeenzymes
involveindegradationofthesecomponents

5. Schematicdiagramofmorphologyandarrangementofsomecellwall
componentsalongwiththerespectiveenzymesresponsibleforthe
degradationofthecellwallcomponents
Hemicellulase
Cellulase
Proteases
Pectate lyase
Proteases
Cellulase
Hemicellulase
Cellulase

6. Cutinase
Cutinis the main component of the cuticle. The upper part of the
cuticle is admixed with waxes, whereas its lower part, in the region
where it merges into the outer walls of epidermal cells, is admixed
with pectin and cellulose.
Many fungi and a few bacteria have been shown to produce
cutinasesand/or nonspecific esterases, i.e., enzymes that can
degrade cutin.
Cutinases break cutin molecules and release monomers (single
molecules) as well as oligomers (small groups of molecules) of the
component fatty acid derivatives from the insoluble cutin polymer.

7. Theinvolvementofcutinaseinthepenetrationofthehost
cuticlebyplantpathogenicfungiisshownbyseveralfacts.For
example,theenzymereachesitshighestconcentrationatthe
penetratingpointofthegermtubeandattheinfectionpegof
appressoriumformingfungi.
Pathogens that produce higher levels of cutinase seem to be
more virulent than others.
e. g. The germinating spores of a virulent isolate of the fungus
Fusarium produced much more cutinase than those of an
avirulent isolate of the same fungus.
The fungus Botrytis cinerea, the cause of numerous types of
diseases on many plants, produces a cutinase and a lipase, both
of which break down cutin.

8. Diagrammaticrepresentationofcuticlepenetrationbya
germinatingfungusspore.Constitutivecutinasereleasesafew
cutinmonomersfromtheplantcuticle.Thesetriggerexpression
ofthecutinasegenesofthefungus,leadingtotheproductionof
morecutinase(s),whichmaceratesthecuticleandallows
penetrationbythefungus.

9. Pectinases
Pectic substances constitute the main components of the middle
lamella, i.e., the intercellular cement that holds in place the cells of
plant tissues.
Pectic substances also make up a large portion of the primary cell
wall in which they form an amorphous gel filling the spaces
between the cellulose microfibrils.
Several enzymes degrade pectic substances and are known as
pectinases or pectolytic enzymes. Some of them, e.g., the pectin
methyl esterases, remove small branches off the pectin chains.
Pectin-degrading enzymes have been shown to be involved in the
production of many fungal and bacterial diseases, particularly
those characterized by the soft rotting of tissues. Various
pathogens produce different sets of pectinases and their isozymes.

10. e.g.
InsomeColletotrichum-causedanthracnoses,thefungus
producesonepectinlyasethatisakeyvirulencefactorindisease
development.
Insomediseases,e.g.,thebacterialwiltofsolanaceouscrops
causedbyRalstoniasolanacearum,pectinolyticenzymes
collectivelyareabsolutelyessentialfordiseasetodevelop,
althoughsomeofthemindividuallyseemtonotberequiredfor
diseasebutratherforacceleratedcolonizationandenhanced
aggressivenessbybacteria.
Pectin-degradingenzymesareproducedandplayaroleinthe
abilityofnematodes,suchastherootknotnematode,Meloidogyne
javanica,forthepenetrationofroottissues,movementbetween
plantcellsalongthemiddlelamella,andpossiblyintheformation
ofteemultinucleategiantcellsonwhichthenematodefeeds
throughouttherestofitslife.

11. Cellulases
Cellulose is also a polysaccharide, but it consists of chains of
glucose (1-4) β-d-glucan molecules. The glucose chains are held to
one another by a large number of hydrogen bonds. Cellulose
occurs in all higher plants as the skeletal substance of cell walls in
the form of microfibrils.
Theenzymaticbreakdownofcelluloseresultsinthefinal
productionofglucosemolecules.Theglucoseisproducedbya
seriesofenzymaticreactionscarriedoutbyseveralcellulasesand
otherenzymes.
Cellulose-degradingenzymes(cellulases)havebeenshowntobe
producedbyseveralphytopathogenicfungi,bacteria,and
nematodesandareundoubtedlyproducedbyparasitichigher
plants.

12. Saprophyticfungi,mainlycertaingroupsofbasidiomycetes,
and,toalesserdegree,saprophyticbacteriacausethe
breakdownofmostofthecellulosedecomposedinnature.
•Inlivingplanttissues,cellulolyticenzymessecretedby
pathogensplayaroleinthesofteninganddisintegrationofcell
wallmaterial.Theyfacilitatethepenetrationandspreadofthe
pathogeninthehostandcausethecollapseanddisintegration
ofthecellularstructure,therebyaidingthepathogeninthe
productionofdisease.
•Cellulolyticenzymesmayfurtherparticipateindirectlyin
diseasedevelopmentbyreleasing,fromcellulosechains,
solublesugarsthatserveasfoodforthepathogenand,inthe
vasculardiseases,byliberatingintothetranspirationstream
largemoleculesfromcellulose,whichinterferewiththenormal
movementofwater.
•Inthebacterialwiltoftomato,productionofanendocellulase
bythebacteriumwasrequiredforthelattertobepathogenic
andinducethedisease.

13. Hemicellulases
Cross-linkingglycans,knownearlierashemicelluloses,are
complexmixturesofpolysaccharidepolymersthatcan
hydrogen-bondtoandmaycoverandlinkcellulosemicrofibrils
together.
Theircompositionandfrequencyseemtovaryamongplant
tissues,plantspecies,andwiththedevelopmentalstageofthe
plant.
Cross-linkingglycansareamajorconstituentoftheprimary
cellwallandmayalsomakeupavaryingproportionofthe
middlelamellaandsecondarycellwall.
Hemicellulosicpolymersincludeprimarilyxyloglucansand
glucuronoarabinoxylans, but also glucomannans,
galactomannans,arabinogalactans,andothers.

14. Theenzymaticbreakdownofhemicellulosesappearsto
requiretheactivityofmanyenzymes.Severalhemicellulases
seemtobeproducedbymanyplantpathogenicfungi.e.g.Rice
blastfungus,Magnaportheoryae.
Dependingonthemonomerreleasedfromthepolymeron
whichtheyact,theparticularenzymesarecalledxylanase,
galactanase,glucanase,arabinase,mannase,andsoon.e.g.
XylanasessecretedbyBotrytiscinerea.

15. Lignindegradingenymes
Ligninisfoundinthemiddlelamella,aswellasinthesecondary
cellwallofxylemvesselsandthefibersthatstrengthenplants.Itis
alsofoundinepidermalandoccasionallyhypodermalcellwallsof
someplants.Thelignincontentofmaturewoodyplantsvaries
from15to38%andissecondonlytocelluloseinabundance.
Itisgenerallyaccepted,however,thatonlyasmallgroupof
microorganismsiscapableofdegradinglignin.

16. Actually,onlyabout500speciesoffungi,almostallofthem
basidiomycetes,havebeenreportedsofarasbeingcapableof
decomposingwood.Aboutone-fourthofthesefungi(thebrown
rotfungi)seemtocausesomedegradationofligninbutcannot
utilizeit.
e.g.
Mostoftheligninintheworldisdegradedandutilizedbya
groupofbasidiomycetescalledwhiterotfungi.Itappearsthat
whiterotfungisecreteoneormoreenzymes(ligninases),which
enablethemtoutilizelignin.
Inadditiontowood-rottingbasidiomycetes,severalother
pathogens,primarilyseveralascomycetesandimperfectfungi
andevensomebacteria,apparentlyproducesmallamountsof
lignin-degradingenzymesandcausesoftrotcavitiesinwood
theycolonize.

17. Proteases/proteinases/peptidases
Plantcellscontaininnumerabledifferentproteins,whichplay
diverserolesascatalystsofcellularreactions(enzymes)oras
structuralmaterial(inmembranesandcellwalls).Proteinsare
formedbythejoiningtogetherofnumerousmoleculesofabout20
differentkindsofaminoacids.
Allpathogensseemtobecapableofdegradingmanykindsof
proteinmolecules.Theplantpathogenicenzymesinvolvedin
proteindegradationaresimilartothosepresentinhigherplants
andanimalsandarecalledproteasesorproteinasesor,
occasionally,peptidases.
Consideringtheparamountimportanceofproteinsasenzymes,
constituentsofcellmembranes,andstructuralcomponentsof
plantcellwalls,thedegradationofhostproteinsbyproteolytic
enzymessecretedbypathogenscanprofoundlyaffectthe
organizationandfunctionofthehostcells.

18. Amylases
Starch is the main reserve polysaccharide found in plant
cells. Starch is synthesized in the chloroplasts and, in
nonphotosynthetic organs, in the amyloplasts.
Starch is a glucose polymer and exists in two forms:
amylose, an essentially linear molecule, and amylopectin, a
highly branched molecule of various chain lengths.
Most pathogens utilize starch, and other reserve
polysaccharides, in their metabolic activities. The degradation
of starch is brought about by the action of enzymes called
amylases. The end product of starch breakdown is glucose and
it is used by the pathogens directly.

19. Lipases & phospholipases
Various types of lipids occur in all plant cells, with the most
important being phospholipids and glycolipids, both of which,
along with protein, are the main constituents of all plant cell
membranes.
Oils and fats are found in many cells, especially in seeds where
they function as energy storage compounds; wax lipids are found
on most aerial epidermal cells. The common characteristic of all
lipids is that they contain fatty acids, which may be saturated or
unsaturated.

20. Severalfungi,bacteria,andnematodesareknowntobe
capableofdegradinglipids.Lipolyticenzymes,calledlipases,
phospholipases,andsoon,hydrolyzeliberationofthefatty
acidsfromthelipidmolecule.Thefattyacidsarepresumably
utilizedbythepathogendirectly.
Someofthem,beforeorafterhyperoxidationbyplant
lipoxygenasesoractiveoxygenspecies,providesignal
moleculesforthedevelopmentofplantdefensesandalsoact
asantimicrobialcompoundsthatinhibitthepathogendirectly.

21. Pathogenic effects on physiological functions of plants
1. Effect of pathogens on photosynthesis
2. Effect of pathogens on translocations of water and nutrients
in the host plant
a. Interference with upward translocation of water and
inorganic nutrients
b. Effect on absorption of water by roots
c. Effect on translocation of water through the xylem
d. Effect on Transpiration
e. Interference with translocation of organic nutrients
through the phloem
3. Effect of pathogens on host plant respiration
4. Effect of pathogens on permeability of cell membranes
5. Effect of pathogens on transcriptions and translations
6. Effect of pathogens on plant growth
7. Effect of pathogens on plant reproduction

22. 1.Effect of pathogens on photosynthesis
Inleafspot,blight,andotherkindsofdiseasesinwhichthereis
destructionofleaftissue,e.g.,incerealrustsandfungalleaf
spots,bacterialleafspots,viralmosaicsandyellowingand
stuntingdiseasesorindefoliations,photosynthesisisreduced
becausethephotosyntheticsurfaceoftheplantislessened.
Insomefungalandbacterialdiseases,photosynthesisis
reducedbecausethetoxins,suchastentoxinandtabtoxin,
producedbythesepathogensinhibitsomeoftheenzymesthat
areinvolveddirectlyorindirectlyinphotosynthesis.
Inplantsinfectedbymanyvascularpathogens,stomataremain
partiallyclosed,chlorophyllisreduced,andphotosynthesis
stopsevenbeforetheplanteventuallywilts.
Mostvirus,mollicute,andnematodediseasesalsoinduce
varyingdegreesofchlorosisandstunting.Inthemajorityof
suchdiseases,thephotosynthesisofinfectedplantsisreduced
greatly.

23. 2. Effect of pathogens on translocations of water and nutrients in
the host plant
a.Interference with upward translocation of water and inorganic
nutrients
Manyplantpathogensinterfereinoneormorewayswiththe
translocationofwaterandinorganicnutrientsthroughplants.:
Somepathogensaffecttheintegrityorfunctionoftheroots,
causingthemtoabsorblesswater;
Otherpathogens,bygrowinginthexylemvesselsorbyother
means,interferewiththetranslocationofwaterthroughthestem;
and,
Insomediseases,pathogensinterferewiththewatereconomy
oftheplantbycausingexcessivetranspirationthroughtheir
effectsonleavesandstomata.

24. b.Effectonabsorptionofwaterbyroots
Rootinjuryaffectstheamountoffunctioningrootsdirectlyand
decreasesproportionatelytheamountofwaterabsorbedbythe
rootsinmanywayssuchas:
Manypathogens,suchasdamping-offfungi,root-rottingfungi
andbacteria,mostnematodes,andsomeviruses,causean
extensivedestructionoftherootsbeforeanysymptomsappearon
theabovegroundpartsoftheplantandsomebacteriaand
nematodescauserootgallsorrootknots,whichinterferewiththe
normalabsorptionofwaterandnutrientsbytheroots.
Somevascularparasites,alongwiththeirothereffects,seemto
inhibitroothairproduction,whichreduceswaterabsorption.
Theseandotherpathogensalsoalterthepermeabilityofroot
cells,aneffectthatfurtherinterfereswiththenormalabsorptionof
waterbyroots.

25. c. Effect on Translocation of Water through the Xylem
Fungalandbacterialpathogensthatcausedampingoff,stem
rotsandcankersmayreachthexylemvesselsintheareaofthe
infectionandinhibitsthetranslocationofwaterthroughxylem
vessels.
Themosttypicalandcompletedysfunctionofxylemin
translocatingwater,however,isobservedinthevascularwilts
causedbythefungiCeratocystis,Ophiostoma,Fusarium,and
VerticilliumandbacteriasuchasPseudomonas,Ralstonia,and
Erwinia.Invascularwilts,affectedvesselsmaybefilledwiththe
bodiesofthepathogenandwithsubstancessecretedbythe
pathogenorbythehostinresponsetothepathogenandmay
becomeclogged.
CertaingallformingpathogenssuchasAgrobacterium
tumefaciens,Plasmodiophorabrassicae),andMeloidogynesp.,
inducegallformationinthestem,roots,orboth.Theenlarged
andproliferatingcellsnearoraroundthexylemexertpressure
onthexylemvessels,whichmaybecrushedanddislocated,
therebybecominglessefficientintransportingwater.

26. d. Effect on Transpiration
Inplantdiseasesinwhichthepathogeninfectstheleaves,
transpirationisusuallyincreased.Thisistheresultofdestruction
ofatleastpartoftheprotectionaffordedtheleafbythecuticle,an
increaseinthepermeabilityofleafcells,andthedysfunctionof
stomata.
Ifwaterabsorptionandtranslocationcannotkeepupwiththe
excessivelossofwater,lossofturgorandwiltingofleavesfollow.
Thesuctionforcesofexcessivelytranspiringleavesareincreased
abnormallyandmayleadtocollapseordysfunctionofunderlying
vesselsthroughtheproductionoftylosesandgums.Indiseases
suchasrusts,inwhichnumerouspustulesformandbreakupthe
epidermis,inmostleafspots,inwhichthecuticle,epidermis,andall
theothertissues,includingxylem,maybedestroyedintheinfected
areas,inthepowderymildews,inwhichalargeproportionofthe
epidermalcellsareinvadedbythefungus,andinapplescab,in
whichthefungusgrowsbetweenthecuticleandtheepidermis-inall
theseexamples,thedestructionofaconsiderableportionofthe
cuticleandepidermisresultsinanuncontrolledlossofwaterfrom
theaffectedareas.

27. e. Interference with translocation of organic nutrients through the
phloem
Instemdiseasesofwoodyplantsinwhichcankersdevelop,the
pathogenattacksandremainsconfinedtothebarkforaconsiderable
time.Duringthattimethepathogenattacksandmaydestroythe
phloemelementsinthatarea,therebyinterferingwiththedownward
translocationofnutrients.
Indiseasescausedbyphytoplasmas,aswellasindiseasescaused
byphloem-limitedfastidiousbacteria,bacteriaexistandreproducein
thephloemsievetubes,therebyinterferingwiththedownward
translocationofnutrients.
Insomevirusdiseases,particularlytheleaf-curlingtypeandsome
yellowsdiseases,starchaccumulationintheleavesismainlythe
resultofdegeneration(necrosis)ofthephloemofinfectedplants,
whichisoneofthefirstsymptoms.Itisalsopossible,however,at
leastinsomevirusdiseases,thattheinterferencewithtranslocation
ofstarchstemsfrominhibitionbythevirusoftheenzymesthatbreak
downstarchintosmaller,translocatablemolecules

28. 3. Effect of pathogens on host plant respiration
Whenplantsareinfectedbypathogens,therateofrespiration
generallyincreases.Thismeansthataffectedtissuesuseuptheir
reservecarbohydratesfasterthanhealthytissueswould.The
increasedrateofrespirationappearsshortlyafterinfection-
certainlybythetimeofappearanceofvisiblesymptomsand
continuestopathogen.Afterthat,respirationdeclinestonormal
levelsortolevelsevenlowerthanthoseofhealthyplants.
Severalchangesinthemetabolismofthediseasedplant
accompanytheincreaseinrespirationafterinfection.Thus,the
activityorconcentrationofseveralenzymesoftherespiratory
pathwaysseemstobeincreased.
Increasedrespirationindiseasedplantsisalsoaccompaniedby
anincreasedactivationofthepentosepathway,whichisthemain
sourceofphenoliccompounds.
Increasedrespirationissometimesaccompaniedbyconsiderably
morefermentationthanthatobservedinhealthyplants,probably
asaresultofanacceleratedneedforenergyinthediseasedplant
underconditionsinwhichnormalaerobicrespirationcannot
providesufficientenergy.

29. 4. Effect of pathogens on permeability of cell membranes
Membranes function as permeability barriers that allow passage
into a cell only of substances the cell needs and inhibit passage
out of the cell of substances needed by the cell.
Changesincellmembranepermeabilityareoftenthefirst
detectableresponsesofcellstoinfectionbypathogens,tomost
host-specificandseveralnonspecifictoxins,tocertainpathogen
enzymes,andtocertaintoxicchemicals,suchasairpollutants.
Themostcommonlyobservedeffectofchangesincellmembrane
permeabilityisthelossofelectrolytes,.
Disruptionordisturbanceofthecellmembranebychemicalor
physicalfactorsalters(usuallyincreases)thepermeabilityofthe
membranewithasubsequentuncontrollablelossofuseful
substances,aswellastheinabilitytoinhibittheinflowof
undesirablesubstancesorexcessiveamountsofanysubstances.

30. 5. Effect of pathogens on transcriptions and translations
a. Effect on Transcription
Severalpathogens,particularlyvirusesandfungalobligate
parasites,suchasrustsandpowderymildews,affectthe
transcriptionprocessininfectedcells.
Insomecases,pathogensaffecttranscriptionbychangingthe
composition,structure,orfunctionofthechromatinassociated
withthecellDNAandinsomediseases,especiallythosecaused
byviruses,thepathogen,throughitsownenzymeorbymodifying
thehostenzyme(RNApolymerase)thatmakesRNA.
b. Effect on Translation
Infectedplanttissuesoftenhaveincreasedactivityinseveral
enzymesandaffectthe:
Increasesinproteinsynthesisininfectedtissueshavebeen
observedprimarilyinhostsresistanttothepathogenandreach
theirhighestlevelsintheearlystagesofinfection
Muchoftheincreasedproteinsynthesisinplantsattackedby
pathogensreflectstheincreasedproductionofenzymesandother
proteinsinvolvedinthedefensereactionsofplants.

31. 6. Effect of pathogens on plant growth
Pathogensthatdestroypartofthephotosyntheticareaofplants
andcausesignificantlyreducedphotosyntheticoutputoften
resultinsmallergrowthoftheseplantsandsmalleryields.
Pathogensthatdestroypartoftherootsofaplantorclogtheir
xylemorphloemelements,therebyseverelyinterferingwiththe
translocationofwaterandofinorganicororganicnutrientsin
theseplants,oftencauseareductioninsizeandyieldsbythese
plantsand,sometimes,theirdeath.
Inmanyplantdiseases,however,infectedtissuesorentireplants
increaseorreduceabnormallyinsize.
Somecharacteristiceffectsonplantgrowtharecausedbythe
phloeminhabitingphytoplasmasproducingshortandbushy
appearance(witches’brooms).Themostfrequentandunusual
effectsonplantgrowtharethosecausedbyviruses(andviroids)
e.g.Stuntingordwarfingofinfectedplants,whereasothers
causerollingorcurlingofleaves,abnormallyshapedfruit,etc.

32. 7. Effect of pathogens on plant reproduction
Pathogensreducetheplantssize,reduceflowersandfruit
settingandseedswhichareofinferiorvigorandvitality.
Manypathogenshaveadirectadverseeffectonplant
reproductionbecausetheyattackandkilltheflowers,fruit,or
seeddirectly,orinterfereandinhibittheirproduction,
Pathogensinterferewiththereproductionoftheirhost
-byinfectingandkillingtheflowersofthehost.
-bykillingtheembryo,thatwouldhaveproducedtheseed,and
replacingthecontentsoftheseedwithitsownfruiting
structureoritsownspores.
Finally,insomediseasescausedbyviruses,phytoplasmas,
orphloemlimitedbacteria,noflowersareproducedorthose
producedaresterile,andthereforefewornofruitandseedare
produced.