BACKGROUND
magnaporthe oryzae is a fungal plant pathogen which causes devastating diseases on several grass species including the cereals rice, wheat and barley
<cit> .
on rice the disease is known as “rice blast” and it causes the most important economic losses in rice cultivation world-wide.
recently the fungus attracted increased attention because it has become a major problem in barley and wheat cultivation, especially in south america, where the disease is named wheat or head blast
<cit> .
thus, there is an urgent need for novel resistance traits which could be engineered into modern elite barley and wheat cultivars.
as a potential source for these traits, we investigated the nonhost response of barley against non-adapted magnaporthe isolates.
this fairly understood phenomenon protects all barley cultivars from infection with magnaporthe species that are pathogenic on other grasses like digitaria or pennisetum <cit> .
generally, nonhost resistance is regarded as durable in the wild and effective against all genetic variants of a pathogen species
<cit> .
infection of barley with a magnaporthe host or nonhost isolate starts when a three-celled conidium attaches to the barley leaf surface facilitated by formation of spore tip mucilage .
adhesion of the subsequently formed germ tube is also accompanied by secretion of an extracellular matrix.
at the tip of the germ tube a specialized cell, called an appressorium, is formed in which a high turgor is generated that supports mechanical penetration of the underlying epidermal tissue.
the next infection stages are different between magnaporthe host and nonhost isolates.
the host pathogen invades epidermal cells with bulbous hyphae initiating a short biotrophic phase of colonization.
this is followed by a switch to necrotrophy in the course of which the pathogen feeds on actively killed host cells
<cit> .
the disease cycle is completed after sporulation takes place in lesions that become visible on infected leaves.
by contrast, penetration of barley leaves by nonhost isolates is generally counterattacked by active defense reactions that block completion of the pathogen’s life-cycle
<cit> .
these reactions may act either during or after penetration and therefore contribute to the penetration and post-penetration defense repertoire, respectively.
for barley, a typical plant reaction associated with penetration resistance against different magnaporthe species is the accumulation of autofluorescent material at the side of attempted penetration beneath a fungal appressorium which is associated with the formation of a cell wall apposition, a so-called papilla
<cit> .
diminishing the effectiveness of penetration resistance, e.g.
by interfering with the actin cytoskeleton or by using barley mutants, revealed that a hypersensitive reaction of attacked epidermal cells functions as a second line of defense, similar to a backup strategy
<cit> .
the kinetic of the latter post-penetration resistance response suggests that it acts before the fungus switches its life-style to necrotrophy.
while much is known about signal exchange between hosts and pathogens during and after penetration, it is still uncertain whether signaling occurs beforehand at the surface.
for barley the accumulation of novel mrnas was shown at 4 hours after inoculation with the wheat powdery mildew fungus
<cit> .
at this stage of infection the barley nonhost pathogen had not yet formed appressorial germ tubes, however penetration attempts by the primary germ tube could not be excluded.
for rice it was reported that a transcriptional up-regulation of genes takes place at  <dig> hours after inoculation with m. oryzae, a timeframe at which the fungus had formed germ tubes and appressoria
<cit> .
a potential source for signal components which may be released during the early stages of infection is the cuticle which represents the layer of the leaf surface firstly touched by pathogens
<cit> .
the cuticle mainly consists of cutin, a polymer built from c <dig> and c <dig> fatty acid units.
intra-cuticular and epi-cuticular waxes are embedded in the cutin polymer matrix or deposited on its outer surface, respectively
<cit> .
generally, cuticular waxes are a complex mixture of long-chain aliphatic lipids with chain lengths from  <dig> to  <dig> c-atoms, triterpenoids and other cyclic compounds, all of which could be released by solvent extraction
<cit> .
the very long chain fatty acids  are elongated in the epidermal er starting with c <dig> and c <dig> fatty acids.
this elongation is catalyzed by an elongation enzyme-complex consisting of four different enzymes
<cit> .
thereafter the vlfcs are either processed to primary alcohols and wax-esters in the acyl reduction pathway or reduced to alkanes in the decarbonylation pathway .
alkanes might then be further oxidized to secondary alcohols and ketones by the activity of the enzyme mid-chain alkane hydroxylase .
in arabidopsis a cytochrome p <dig> protein of the cyp <dig> gene family, designated cyp96a <dig>  was found to have mah-enzymatic activity
<cit> .
interestingly, inflorescence stems of all arabidopsis mah <dig> mutant lines were covered with a visually normal wax surface although they were devoid of secondary alcohols and ketones in comparison to the wild-type suggesting a high grade of plasticity in the formation of wax layers.
here, we report on a barley cytochrome p <dig> gene which shows greatest homology to cyp <dig> family members.
the gene was identified in a large transcriptome analysis to be specifically up-regulated after inoculation of barley plants with a magnaporthe nonhost isolate
<cit> .
our results indicate that the gene was expressed in response to the ectoparasitic growth of the pathogen on the leaf surface and functional gene silencing analysis suggests its involvement in execution of penetration resistance.
RESULTS
infection of barley with a magnaporthe nonhost isolate induces expression of cyp96b22
in a previous macroarray-based study, we had identified the barley ipk cr-est clone ho07g <dig> to be among the three genes with the highest differential transcript abundance in the barley-magnaporthe nonhost interaction as compared to host interaction
<cit> .
a blastn search against the high confidence sequences of the recently published barley genome identified ho07g <dig> as part of the mloc_ <dig>  cdna .
the amino acid sequence deduced from ho07g <dig> is almost identical to the c-terminus of the mloc_ <dig>  protein.
differences led to three single amino acid changes in the deduced proteins which might be due to differences among cultivars used.
the mloc_ <dig>  protein is annotated as a cytochrome p <dig>  family protein.
a search for conserved domains within the entire  <dig>  kda protein deduced from mloc_ <dig>  revealed a transmembrane region and a mid-chain hydroxylase cyp domain.
a phylogenetic analysis of the protein with rice cyp sequences deposited at the cytochrome p <dig> homepage
<cit>  grouped the protein within the cyp <dig> family of the cyp <dig> clan .
the sequence shares 67% identity to cyp96b <dig> of brachypodium distachyon and 59% identity to cyp96b <dig> of rice .
as suggested in bak et al.
<cit> , the sequence was sent to david nelson, cyp nomenclature committee, who named it cyp96b <dig>
<cit>  and used as a query for a homology search of the barley cyp sequence designated cyp96b <dig>  the phylogenetic analysis was performed on the phylogeny.fr platform
<cit>  using the maximum likelihood method implemented in phyml.
the scale bar indicates  <dig>  substitution per site, branch support values are given in percent.
to simplify the tree view only members of the rice cyp <dig> family and selected representatives of the other families in the cyp <dig> clan and additional cyp clans are shown.
ectoparasitic growth of magnaporthe on barley induces expression of cyp96b22
at first, we confirmed the results obtained in the macroarray study on the differential expression pattern of cyp96b <dig> in response to infection with different magnaporthe isolates in independent experiments.
therefore, barley cultivar vada was inoculated with the m. oryzae host isolate  which resulted in clearly visible disease symptoms on primary leaves at five days after inoculation .
at the same time, leaves were inoculated with a pennisetum-infecting nonhost isolate  and, as expected, did not show any disease symptoms even after a prolonged incubation period .
according to zellerhoff et al.
<cit>  and faivre-rampant et al.
<cit>  both isolates are most likely members of different magnaporthe species.
for rt-qpcr analysis the epidermis from infected leaves of the same experiments was peeled .
cyp96b <dig> expression in mock treated samples was higher at early time points, i.e.
at  <dig> and 12 h p.i.
and decreased thereafter .
essentially transcript profiles of cyp96b <dig> after inoculation with the host or nonhost isolate followed the same pattern.
however at 6 h p.i.
the magnitude of expression was significantly higher in the host as compared to the mock situation and even higher in the nonhost interaction .
thereby our data perfectly resembled the previously published results on the differential expression of cyp96b <dig> <cit> .
the observed early induction of cyp96b <dig> expression after magnaporthe inoculation raised the question of the stimulus which caused this response.
to discern the cellular events taking place during the early barley-magnaporthe interaction transgenic, fluorescent-labeled magnaporthe isolates were generated for both the host and nonhost isolate and inoculated onto barley plants.
analysis using epifluorescence microscopy revealed that at 6 h p.i.
the majority of conidiospores were in the stage of germination while only a few already had formed appressoria .
penetration pegs or invasive hyphae inside attacked epidermal cells were not observed before 24 h p.i either in the host or the nonhost interaction.
it was concluded, therefore, that under our experimental conditions penetration took place between  <dig> and 24 h p.i.
this, in turn, led to the conclusion that cyp96b <dig> transcript accumulation at 6 h p.i.
was triggered during the ectoparasitic growth of magnaporthe on the surface of barley leaves and possibly before penetration.
cyp96b <dig> expression caused by m. oryzae does not require penetration
to further test the hypothesis that cyp96b <dig> expression in barley is triggered before penetration, we made use of a fungicidal treatment and a penetration deficient m. oryzae mutant which prevent pathogen invasion after or before appressorium formation, respectively.
the fungicide beam has the active ingredient tricyclazole which inhibits the reduction of  <dig> ,8-trihydroxynaphtalene to vermelone in the melanin biosynthetic pathway
<cit> .
the absence of melanin then leads to magnaporthe appressoria unable to penetrate and beam is therefore widely used in rice cultivation to control blast disease.
here, we applied beam by soil drench to barley plants prior to inoculation with magnaporthe host or nonhost isolates.
beam-treated plants showed significantly less disease symptoms after inoculation with the m. oryzae host isolate th <dig> in comparison to the respective control indicating the effectiveness of the fungicide .
microscopic analysis revealed that both the host and nonhost isolate developed infection structures on the leaf surface of beam-treated plants up to the stage of appressorium formation .
thereafter, as anticipated, beam-treatment led to the absence of dark-pigmentation, i.e.
melanization, in appressoria which was found for approximately half of all incidences.
almost no penetration pegs or invasive hyphae were found in epidermal cells of beam-treated plants.
it was concluded, therefore, that even in those cases in which dark pigmentation of appressoria was observed the fungus was mostly unable to penetrate .
the observation of few cells in which invasive hyphae were detected in the host interaction is consistent with the rare disease symptoms observed on the beam-treated plants  and indicated that the fungicide concentration used in our experiments did not mediate a 100% protection.
despite the blocked penetration on beam-treated plants, cyp96b <dig> transcripts did still accumulate at 6 h p.i.
in response to inoculation with the host or nonhost isolate and in comparison to the respective control .
to further confirm this observation, we made use of the m. oryzae mutant Δpmk <dig> which lacks a functional conserved mitogen-activated protein kinase
<cit> .
this mutant cannot form normal appressoria and is therefore unable to penetrate rice or barley leaves.
since the mutant was generated in the genetic background of m. oryzae isolate guy <dig>  we firstly verified that guy <dig> resembled the infection phenotype of isolate th <dig> on barley cv.
vada .
in agreement with xu and hamer
<cit> , Δpmk <dig> was found to be apathogenic on barley .
similarly, microscopic evaluation confirmed the results of these authors by showing that the mutant only formed melanin-less, swollen structures instead of mature appressoria at the tip of their germ tubes .
barley epidermis inoculated with Δpmk <dig> was harvested in the same time-course experiments as for the beam approach and rt-qpcr experiments showed enhanced expression of cyp96b <dig> at 6 h p.i.
.
taken together our experiments with beam and the Δpmk <dig> mutant undoubtedly revealed that magnaporthe triggers cyp96b <dig> expression in barley independent from appressorium formation or successful penetration in both the host and nonhost interaction.
for comparison the expression of pr1b was monitored in the same set of rt-qpcr samples.
in accordance with results published by jarosch et al.
<cit> , pr1b transcripts strongly accumulated in barley leaves in response to inoculation with the magnaporthe host isolate .
by contrast, this strong pr1b transcript accumulation was neither seen in beam-treated plants nor in response to the inoculation with the Δpmk <dig> mutant , indicating that induction of pr1b expression depends on the formation of mature appressoria and/or successful penetration.
knock-down of cyp96b <dig> expression led to compromised penetration resistance
our next aim was to functionally evaluate whether cyp96b <dig> has a crucial role in the defense of barley against different magnaporthe isolates.
for this purpose, we used a transient barley stripe mosaic virus -induced gene silencing assay which was first described by holzberg et al.
<cit>  and which we had already adapted for magnaporthe infection assays
<cit> .
we inserted a 234 bp fragment of the cyp96b <dig> coding sequence into the γ-subunit of bsmv.
using the si-fi software, this construct was predicted to lead to efficient degradation of the cyp96b <dig> mrna and not to target transcripts of other barley genes  .
infection of barley plants with bsmv exhibiting the silencing construct  or without it  was done on primary leaves.
typical, mosaic-like, viral disease symptoms arose on 10-30% of individuals of both groups at 10–14 days after inoculation with bsmv, indicative of a successful virus infection.
the third leaves of plants showing virus symptoms were cut off and divided into three equally long pieces .
two of them were inoculated with magnaporthe isolate cd <dig> and one with th <dig> in a detached leaf assay.
since previous studies showed that the knock-down of gene expression in bsmv-vigs experiments can vary between different plants, silencing efficiency was monitored by rt-qpcr using leaf material harvested 24 h after inoculation with the nonhost isolate .
on average, the expression level of cyp96b <dig> was lower in plants inoculated with the bsmv-γcyp than in bsmv-γmcsnew-infected control plants.
for microscopic evaluation plants were grouped , thereby one group consisted of the five plants inoculated with bsmv without silencing construct  and the other encompassed those five plants showing the lowest cyp96b <dig> transcript abundance after inoculation with bsmv with silencing construct .
in the latter group transcript abundance was reduced by 86% as compared to plants of the first group .
at least  <dig> infection sites were screened for each plant from both groups and assigned to categories reflecting typical plant defense responses .
above that, the presence of invasive hyphae in an attacked epidermal cell was recorded for each infection site.
in the nonhost interaction with isolate cd <dig>  the vast majority of attacked epidermal cells in the group of plants without silencing showed a local accumulation of autofluorescent material beneath appressoria associated with the formation of a cell wall apposition called papilla .
invasive hyphae were never observed in these cells indicating an efficient defense response.
silencing of cyp96b <dig> led to a significant reduction of infection sites grouped in this category.
instead a significant higher frequency of attacked epidermal cells was found showing autofluorescence of the entire cell wall which was verified in two independent replicate experiments.
according to zellerhoff et al.
<cit>  this interaction category could be interpreted as post-penetration plant defense response.
this was concluded from a time course experiment where the authors showed that, after successful penetration of ineffective papillae, invasive hyphae of magnaporthe nonhost isolates were ultimately arrested in epidermal cells showing autofluorescent cell walls.
the frequency of infection sites grouped within the other two categories did not differ significantly between plants of both groups .
in the host situation no significant differences were observed among frequencies for each category between plants with or without efficient cyp96b22-silencing .
however, monitoring the invasion success, cyp96b22-silencing led to a higher frequency of invasive hyphae at infection sites where the fungus had successfully penetrated papillae .
in a biological replicate, we observed for the host interaction that cyp96b22-silencing also affected the transition of fungal development from epidermal to mesophyll tissue.
this was evidenced by microscopy of infection stages at which the fungus had already built invasive hyphae in more than 35% of attacked epidermal cells and which was correlated with significant more collapsed mesophyll tissue in plants with efficient cyp96b22-silencing in comparison to control plants .
the collapse of mesophyll tissue is an indication for advanced m. oryzae colonization
<cit> .
to investigate whether the effect of cyp96b22-silencing on enhanced penetration success also applies for other pathogens, vigs experiments were conducted with the barley powdery mildew fungus blumeria graminis f. sp.
hordei .
in this case, the group of plants chosen for microscopic evaluation showed 79% less transcript abundance for cyp96b <dig> in comparison to the group of plants inoculated with bsmv without silencing construct .
however, substantial differences were observed neither among the different categories of plant defense reactions nor in the frequency of successfully formed haustoria.
<dig>  in this case the average silencing efficiency was 79%.
plants of both pools were inoculated with blumeria graminis f. sp.
hordei  in a detached leaf assay.
infection sites were categorized according to defense responses associated with the deposition of autofluorescent material.
for each leaf  <dig> infection sites were evaluated at 48 h after inoculation and the mean and standard error of four leaves are presented.
attacked epidermal cells were further investigated for the presence of mature haustoria  and the frequencies are displayed by shading of bars.
cells found with haustoria were summed up as shown in a separate diagram in the upper right corner.
each category of plant defense responses was tested for significant differences between plants with and without silencing using t-test  and this revealed no significant differences.
it was confirmed in an independent experiment that cyp96b <dig> silencing had no effect on the frequency of haustoria.
discussion
during recent years, large transcriptome profiling has become a routine analysis in the study of plant responses against abiotic and biotic stresses and was considered as a promising tool on the way to improve crop performance
<cit> .
here, we report on a cytochrome p <dig> gene of barley which was identified in a macroarray-based study as differentially regulated during nonhost and host interactions with fungal isolates of the magnaporthe species complex.
we provide evidence that this gene is involved in execution of penetration resistance as an early response to the ectoparasitic growth of the pathogen on the leaf surface.
starting in  <dig>  the leibniz institute of plant genetics and crop plant research  at gatersleben developed a macroarray on the basis of expressed sequence tags  and made it available as a resource for the barley community to study gene expression in response to various treatments
<cit> .
using this array, we identified a set of barley genes whose transcripts accumulate to a higher dose or at earlier time points after inoculation with a magnaporthe nonhost as compared to a magnaporthe host isolate
<cit> .
a prominent member among these genes was an est clone identical to the barley mloc_ <dig> cdna which has a domain characteristic for cytochrome p <dig> enzymes .
plant p450s are involved in primary and secondary metabolic processes and catalyze different monooxygenation and hydroxylation reactions
<cit> .
the wide distribution of p450s in a plant species-specific manner implies that they are subject to high evolutionary pressure which led to diversification and finally to novel metabolic pathways and products with acquired new biological functions.
search for homology among plant p450s placed the mloc_ <dig> protein into the cyp <dig> family  and suggested an enzymatic function as midchain alkane hydroxylase .
the closest arabidopsis homologue of cyp96b <dig> is cyp96a <dig> , which was shown to be a splicing variant of a monocistronic transcript, forming a dimer with cyp96a9
<cit> .
it was suggested that the dimerization is required for the modification of hydrophobic substrates
<cit> .
in arabidopsis cyp96a <dig> transcripts were found less abundant than cyp96a <dig> transcripts
<cit> , accounting for cyp96a <dig> concentration as a rate limiting factor at least at the level of transcriptional regulation.
assuming a similar situation for barley, this could be interpreted as if cyp96b <dig> has gained a novel regulatory function in the resistance response of barley against magnaporthe nonhost isolates.
in this scenario, the transcriptional up-regulation of cyp96b <dig> will lead to enhanced formation of heterodimers with a putative barley protein homologous to cyp96a <dig> and finally to more production of a hydrophobic product.
so far the only cyp <dig> enzyme characterized in more detail is arabidopsis mah <dig> which provides secondary alcohols and ketones as building blocks for the cuticular wax layer .
this outermost surface layer is the first barrier which plant pathogens have to cope with during infection
<cit> .
additionally, the cuticle might also be a reservoir for the release of signal molecules which could be perceived by the attacked plant itself as damage-associated molecular patterns  leading to the execution of defense responses.
alternatively, cuticle constituents could be used by pathogens as host identification factors which was shown e.g.
in the barley-powdery mildew interaction for a c26-aldehyde
<cit> .
cuticle components may also function in bacterial pathogenicity, e.g.
it was shown that the arabidopsis cyp86a <dig>  a plant p <dig> which is involved in cuticle development, is required for the induction of the bacterial type iii secretion system
<cit> .
on the other hand it was shown that free cutin monomers, most likely released by the activity of fungal derived cutinases, may act as endogenous damps and induce plant defense responses
<cit> .
in arabidopsis the main epicuticular wax components are alkanes, secondary alcohols and ketones with a chain length of c <dig>  all of which are synthesized via a mah1-dependent pathway
<cit> .
however, barley cuticles mainly contain a c26-primary alcohol, n-hexacosanol, whose synthesis does not require a mah <dig> enzymatic conversion
<cit> .
it might be, therefore, that mah1-derived wax components in barley are less important against pathogen attack as structural barriers but instead act in signaling.
we addressed the biological function of cyp96b <dig> in a transient assay using bsmv as a tool to knock-down gene expression .
the usefulness of bsmv-mediated vigs for functional genomics in wheat and barley has been demonstrated in several recent publications
<cit>  and we already adopted the assay for the study of barley-magnaporthe interactions
<cit> .
since it was shown for wheat that bsmv-infection prior to inoculation with m. oryzae host isolates may lead to decreased penetration
<cit> , we used in all of our vigs-experiments plants inoculated with bsmv without silencing construct as an internal control .
doing so and comparing these control plants with those showing the highest reduction in cyp96b <dig> transcript abundance, a significant decrease in the frequency of papillae at sites of attempted penetration by the magnaporthe nonhost isolate cd <dig> was observed .
since this latter result went along with more epidermal cells showing deposition of autofluorescent material at entire cell walls, this is indicative of ineffective papillae which could not block magnaporthe penetration
<cit> .
the more rapid transition of fungal invasion across cell walls became even more obvious in the barley host interaction with m. oryzae isolate th <dig> where cyp96b <dig> silencing led to an increase of invasive hyphae in attacked epidermal cells.
similar findings for decreased penetration defense and more frequent entry of pathogens into epidermal cells, were also reported for arabidopsis pen-mutants .
in the light of this finding cyp96b <dig> could be regarded as being a barley pen-gene.
however, neither in arabidopsis pen-mutants nor in our experiments with cyp96b22-vigs, nonhost pathogens were able to sporulate, indicating effective post-penetration resistance mechanisms.
disabling this second line of defense by blocking the eds1-pad4-sag <dig> signaling complex renders arabidopsis fully susceptible even to non-adapted pathogens
<cit> .
in barley, the deposition of autofluorescent material seems to be a cytological marker for this back-up strategy in interactions with magnaporthe.
nonhost resistance is generally considered to be determined by several quantitative trait loci all of which must act in common to prevent infection by a would-be pathogen
<cit> .
only the detailed monitoring of minor cytological changes in bsmv-vigs treated barley plants enabled us to assign a function in penetration resistance against magnaporthe nonhost isolates to the barley gene cyp96b <dig>  it would be advisable, therefore, to refrain from macroscopic evaluation in nonhost analysis after knock-out or knock-down of candidate gene expression.
since the infection process of the adapted powdery mildew fungus on barley plants after knock-down of cyp96b <dig> expression was not altered , we could rule out the possibility of an entire collapse of penetration resistance in these plants.
the finding that cyp96b <dig> may act differently in barley powdery mildew vs. magnaporthe interactions was not astonishing, since similar observations had been made before for barley mlo or rop genes
<cit> .
this ambivalence in gene regulation was interpreted as part of a dedicated response of barley to biotrophic or hemi-biotrophic pathogens.
interestingly, cyp96b <dig> was found to be down-regulated in barley after inoculation with puccinia triticina and puccinia hordei <cit> .
however a functional verification of this result e.g.
in a bsmv-vigs assay has not been reported yet.
an unexpected finding of the present study was the sensing of the ectoparasitic growth of magnaporthe germ tubes at the leaf surface by barley plants.
using the m. oryzae mutant Δpmk <dig>  we undoubtedly verified that penetration is not necessary to trigger cyp96b <dig> expression .
we showed also for magnaporthe nonhost isolates, for which no Δpmk <dig> mutants existed, that they were able to cause the same phenotype by using a fungicide leading to non-functional appressoria and thereby preventing penetration .
other studies also indicated a signal exchange between pathogens and plants before penetration, however the chemical nature of the signal itself was never elucidated
<cit> .
most recently it was shown for arabidopsis that effectors of colletotrichum higginsianum are secreted before penetration
<cit> .
the homology of cyp96b <dig> to mah <dig> enzymes and its implication in synthesis of cuticular waxes leads us to speculate that mah1-dependent waxy components might be deliberated during the mucilage-facilitated attachment of spores and germ tubes to the leaf surface.
thereafter they are sensed as damps and lead to increased defense responses associated with penetration resistance as e.g.
the formation of effective papilla.
in the host situation, m. oryzae isolates might then secrete effector molecules which negate these defense reactions
<cit> .
CONCLUSIONS
our results provide functional evidence that the barley cytochrome p <dig> gene cyp96b <dig> plays a crucial role in execution of the nonhost response of barley against pathogens from the magnaporthe species complex.
in the light of head blast as becoming a more prevalent disease in barley and wheat cultivation, our study might provide useful information for further breeding strategies.
however, more studies are needed to establish whether the deduced protein functions in strengthening of structural barriers against pathogen attack or in generation of signal molecules.

 magnaporthe oryzae is a fungal plant pathogen which causes devastating diseases on several grass species including the cereals rice, wheat and barley. on rice the disease is known as “rice blast” and it causes the most important economic losses in rice cultivation world-wide. recently the fungus attracted increased attention because it has become a major problem in barley and wheat cultivation, especially in south america, where the disease is named wheat or head blast. thus, there is an urgent need for novel resistance traits which could be engineered into modern elite barley and wheat cultivars. as a potential source for these traits, we investigated the nonhost response of barley against non-adapted magnaporthe isolates. this fairly understood phenomenon protects all barley cultivars from infection with magnaporthe species that are pathogenic on other grasses like digitaria or pennisetum. generally, nonhost resistance is regarded as durable in the wild and effective against all genetic variants of a pathogen species. infection of barley with a magnaporthe host or nonhost isolate starts when a three-celled conidium attaches to the barley leaf surface facilitated by formation of spore tip mucilage. adhesion of the subsequently formed germ tube is also accompanied by secretion of an extracellular matrix. at the tip of the germ tube a specialized cell, called an appressorium, is formed in which a high turgor is generated that supports mechanical penetration of the underlying epidermal tissue. the next infection stages are different between magnaporthe host and nonhost isolates. the host pathogen invades epidermal cells with bulbous hyphae initiating a short biotrophic phase of colonization. this is followed by a switch to necrotrophy in the course of which the pathogen feeds on actively killed host cells. the disease cycle is completed after sporulation takes place in lesions that become visible on infected leaves. by contrast, penetration of barley leaves by nonhost isolates is generally counterattacked by active defense reactions that block completion of the pathogen’s life-cycle. these reactions may act either during or after penetration and therefore contribute to the penetration and post-penetration defense repertoire, respectively. for barley, a typical plant reaction associated with penetration resistance against different magnaporthe species is the accumulation of autofluorescent material at the side of attempted penetration beneath a fungal appressorium which is associated with the formation of a cell wall apposition, a so-called papilla. diminishing the effectiveness of penetration resistance, e.g. by interfering with the actin cytoskeleton or by using barley mutants, revealed that a hypersensitive reaction of attacked epidermal cells functions as a second line of defense, similar to a backup strategy. the kinetic of the latter post-penetration resistance response suggests that it acts before the fungus switches its life-style to necrotrophy. while much is known about signal exchange between hosts and pathogens during and after penetration, it is still uncertain whether signaling occurs beforehand at the surface. for barley the accumulation of novel mrnas was shown at 4 hours after inoculation with the wheat powdery mildew fungus. at this stage of infection the barley nonhost pathogen had not yet formed appressorial germ tubes, however penetration attempts by the primary germ tube could not be excluded. for rice it was reported that a transcriptional up-regulation of genes takes place at hours after inoculation with m. oryzae, a timeframe at which the fungus had formed germ tubes and appressoria. a potential source for signal components which may be released during the early stages of infection is the cuticle which represents the layer of the leaf surface firstly touched by pathogens. the cuticle mainly consists of cutin, a polymer built from c and c fatty acid units. intra-cuticular and epi-cuticular waxes are embedded in the cutin polymer matrix or deposited on its outer surface, respectively. generally, cuticular waxes are a complex mixture of long-chain aliphatic lipids with chain lengths from to c-atoms, triterpenoids and other cyclic compounds, all of which could be released by solvent extraction. the very long chain fatty acids are elongated in the epidermal er starting with c and c fatty acids. this elongation is catalyzed by an elongation enzyme-complex consisting of four different enzymes. thereafter the vlfcs are either processed to primary alcohols and wax-esters in the acyl reduction pathway or reduced to alkanes in the decarbonylation pathway. alkanes might then be further oxidized to secondary alcohols and ketones by the activity of the enzyme mid-chain alkane hydroxylase. in arabidopsis a cytochrome p protein of the cyp gene family, designated cyp96a was found to have mah-enzymatic activity. interestingly, inflorescence stems of all arabidopsis mah mutant lines were covered with a visually normal wax surface although they were devoid of secondary alcohols and ketones in comparison to the wild-type suggesting a high grade of plasticity in the formation of wax layers. here, we report on a barley cytochrome p gene which shows greatest homology to cyp family members. the gene was identified in a large transcriptome analysis to be specifically up-regulated after inoculation of barley plants with a magnaporthe nonhost isolate. our results indicate that the gene was expressed in response to the ectoparasitic growth of the pathogen on the leaf surface and functional gene silencing analysis suggests its involvement in execution of penetration resistance.  infection of barley with a magnaporthe nonhost isolate induces expression of cyp96b22 in a previous macroarray-based study, we had identified the barley ipk cr-est clone ho07g to be among the three genes with the highest differential transcript abundance in the barley-magnaporthe nonhost interaction as compared to host interaction. a blastn search against the high confidence sequences of the recently published barley genome identified ho07g as part of the mloc_ cdna. the amino acid sequence deduced from ho07g is almost identical to the c-terminus of the mloc_ protein. differences led to three single amino acid changes in the deduced proteins which might be due to differences among cultivars used. the mloc_ protein is annotated as a cytochrome p family protein. a search for conserved domains within the entire  kda protein deduced from mloc_ revealed a transmembrane region and a mid-chain hydroxylase cyp domain. a phylogenetic analysis of the protein with rice cyp sequences deposited at the cytochrome p homepage grouped the protein within the cyp family of the cyp clan. the sequence shares 67% identity to cyp96b of brachypodium distachyon and 59% identity to cyp96b of rice. as suggested in bak et al., the sequence was sent to david nelson, cyp nomenclature committee, who named it cyp96b and used as a query for a homology search of the barley cyp sequence designated cyp96b the phylogenetic analysis was performed on the phylogeny.fr platform using the maximum likelihood method implemented in phyml. the scale bar indicates substitution per site, branch support values are given in percent. to simplify the tree view only members of the rice cyp family and selected representatives of the other families in the cyp clan and additional cyp clans are shown. ectoparasitic growth of magnaporthe on barley induces expression of cyp96b22 at first, we confirmed the results obtained in the macroarray study on the differential expression pattern of cyp96b in response to infection with different magnaporthe isolates in independent experiments. therefore, barley cultivar vada was inoculated with the m. oryzae host isolate which resulted in clearly visible disease symptoms on primary leaves at five days after inoculation. at the same time, leaves were inoculated with a pennisetum-infecting nonhost isolate and, as expected, did not show any disease symptoms even after a prolonged incubation period. according to zellerhoff et al. and faivre-rampant et al. both isolates are most likely members of different magnaporthe species. for rt-qpcr analysis the epidermis from infected leaves of the same experiments was peeled. cyp96b expression in mock treated samples was higher at early time points, i.e. at and 12 h p.i. and decreased thereafter. essentially transcript profiles of cyp96b after inoculation with the host or nonhost isolate followed the same pattern. however at 6 h p.i. the magnitude of expression was significantly higher in the host as compared to the mock situation and even higher in the nonhost interaction. thereby our data perfectly resembled the previously published results on the differential expression of cyp96b. the observed early induction of cyp96b expression after magnaporthe inoculation raised the question of the stimulus which caused this response. to discern the cellular events taking place during the early barley-magnaporthe interaction transgenic, fluorescent-labeled magnaporthe isolates were generated for both the host and nonhost isolate and inoculated onto barley plants. analysis using epifluorescence microscopy revealed that at 6 h p.i. the majority of conidiospores were in the stage of germination while only a few already had formed appressoria. penetration pegs or invasive hyphae inside attacked epidermal cells were not observed before 24 h p.i either in the host or the nonhost interaction. it was concluded, therefore, that under our experimental conditions penetration took place between and 24 h p.i. this, in turn, led to the conclusion that cyp96b transcript accumulation at 6 h p.i. was triggered during the ectoparasitic growth of magnaporthe on the surface of barley leaves and possibly before penetration. cyp96b expression caused by m. oryzae does not require penetration to further test the hypothesis that cyp96b expression in barley is triggered before penetration, we made use of a fungicidal treatment and a penetration deficient m. oryzae mutant which prevent pathogen invasion after or before appressorium formation, respectively. the fungicide beam has the active ingredient tricyclazole which inhibits the reduction of,8-trihydroxynaphtalene to vermelone in the melanin biosynthetic pathway. the absence of melanin then leads to magnaporthe appressoria unable to penetrate and beam is therefore widely used in rice cultivation to control blast disease. here, we applied beam by soil drench to barley plants prior to inoculation with magnaporthe host or nonhost isolates. beam-treated plants showed significantly less disease symptoms after inoculation with the m. oryzae host isolate th in comparison to the respective control indicating the effectiveness of the fungicide. microscopic analysis revealed that both the host and nonhost isolate developed infection structures on the leaf surface of beam-treated plants up to the stage of appressorium formation. thereafter, as anticipated, beam-treatment led to the absence of dark-pigmentation, i.e. melanization, in appressoria which was found for approximately half of all incidences. almost no penetration pegs or invasive hyphae were found in epidermal cells of beam-treated plants. it was concluded, therefore, that even in those cases in which dark pigmentation of appressoria was observed the fungus was mostly unable to penetrate. the observation of few cells in which invasive hyphae were detected in the host interaction is consistent with the rare disease symptoms observed on the beam-treated plants and indicated that the fungicide concentration used in our experiments did not mediate a 100% protection. despite the blocked penetration on beam-treated plants, cyp96b transcripts did still accumulate at 6 h p.i. in response to inoculation with the host or nonhost isolate and in comparison to the respective control. to further confirm this observation, we made use of the m. oryzae mutant Δpmk which lacks a functional conserved mitogen-activated protein kinase. this mutant cannot form normal appressoria and is therefore unable to penetrate rice or barley leaves. since the mutant was generated in the genetic background of m. oryzae isolate guy we firstly verified that guy resembled the infection phenotype of isolate th on barley cv. vada. in agreement with xu and hamer, Δpmk was found to be apathogenic on barley. similarly, microscopic evaluation confirmed the results of these authors by showing that the mutant only formed melanin-less, swollen structures instead of mature appressoria at the tip of their germ tubes. barley epidermis inoculated with Δpmk was harvested in the same time-course experiments as for the beam approach and rt-qpcr experiments showed enhanced expression of cyp96b at 6 h p.i.. taken together our experiments with beam and the Δpmk mutant undoubtedly revealed that magnaporthe triggers cyp96b expression in barley independent from appressorium formation or successful penetration in both the host and nonhost interaction. for comparison the expression of pr1b was monitored in the same set of rt-qpcr samples. in accordance with results published by jarosch et al., pr1b transcripts strongly accumulated in barley leaves in response to inoculation with the magnaporthe host isolate. by contrast, this strong pr1b transcript accumulation was neither seen in beam-treated plants nor in response to the inoculation with the Δpmk mutant, indicating that induction of pr1b expression depends on the formation of mature appressoria and/or successful penetration. knock-down of cyp96b expression led to compromised penetration resistance our next aim was to functionally evaluate whether cyp96b has a crucial role in the defense of barley against different magnaporthe isolates. for this purpose, we used a transient barley stripe mosaic virus -induced gene silencing assay which was first described by holzberg et al. and which we had already adapted for magnaporthe infection assays. we inserted a 234 bp fragment of the cyp96b coding sequence into the γ-subunit of bsmv. using the si-fi software, this construct was predicted to lead to efficient degradation of the cyp96b mrna and not to target transcripts of other barley genes. infection of barley plants with bsmv exhibiting the silencing construct or without it was done on primary leaves. typical, mosaic-like, viral disease symptoms arose on 10-30% of individuals of both groups at 10–14 days after inoculation with bsmv, indicative of a successful virus infection. the third leaves of plants showing virus symptoms were cut off and divided into three equally long pieces. two of them were inoculated with magnaporthe isolate cd and one with th in a detached leaf assay. since previous studies showed that the knock-down of gene expression in bsmv-vigs experiments can vary between different plants, silencing efficiency was monitored by rt-qpcr using leaf material harvested 24 h after inoculation with the nonhost isolate. on average, the expression level of cyp96b was lower in plants inoculated with the bsmv-γcyp than in bsmv-γmcsnew-infected control plants. for microscopic evaluation plants were grouped, thereby one group consisted of the five plants inoculated with bsmv without silencing construct and the other encompassed those five plants showing the lowest cyp96b transcript abundance after inoculation with bsmv with silencing construct. in the latter group transcript abundance was reduced by 86% as compared to plants of the first group. at least infection sites were screened for each plant from both groups and assigned to categories reflecting typical plant defense responses. above that, the presence of invasive hyphae in an attacked epidermal cell was recorded for each infection site. in the nonhost interaction with isolate cd the vast majority of attacked epidermal cells in the group of plants without silencing showed a local accumulation of autofluorescent material beneath appressoria associated with the formation of a cell wall apposition called papilla. invasive hyphae were never observed in these cells indicating an efficient defense response. silencing of cyp96b led to a significant reduction of infection sites grouped in this category. instead a significant higher frequency of attacked epidermal cells was found showing autofluorescence of the entire cell wall which was verified in two independent replicate experiments. according to zellerhoff et al. this interaction category could be interpreted as post-penetration plant defense response. this was concluded from a time course experiment where the authors showed that, after successful penetration of ineffective papillae, invasive hyphae of magnaporthe nonhost isolates were ultimately arrested in epidermal cells showing autofluorescent cell walls. the frequency of infection sites grouped within the other two categories did not differ significantly between plants of both groups. in the host situation no significant differences were observed among frequencies for each category between plants with or without efficient cyp96b22-silencing. however, monitoring the invasion success, cyp96b22-silencing led to a higher frequency of invasive hyphae at infection sites where the fungus had successfully penetrated papillae. in a biological replicate, we observed for the host interaction that cyp96b22-silencing also affected the transition of fungal development from epidermal to mesophyll tissue. this was evidenced by microscopy of infection stages at which the fungus had already built invasive hyphae in more than 35% of attacked epidermal cells and which was correlated with significant more collapsed mesophyll tissue in plants with efficient cyp96b22-silencing in comparison to control plants. the collapse of mesophyll tissue is an indication for advanced m. oryzae colonization. to investigate whether the effect of cyp96b22-silencing on enhanced penetration success also applies for other pathogens, vigs experiments were conducted with the barley powdery mildew fungus blumeria graminis f. sp. hordei. in this case, the group of plants chosen for microscopic evaluation showed 79% less transcript abundance for cyp96b in comparison to the group of plants inoculated with bsmv without silencing construct. however, substantial differences were observed neither among the different categories of plant defense reactions nor in the frequency of successfully formed haustoria. in this case the average silencing efficiency was 79%. plants of both pools were inoculated with blumeria graminis f. sp. hordei in a detached leaf assay. infection sites were categorized according to defense responses associated with the deposition of autofluorescent material. for each leaf infection sites were evaluated at 48 h after inoculation and the mean and standard error of four leaves are presented. attacked epidermal cells were further investigated for the presence of mature haustoria and the frequencies are displayed by shading of bars. cells found with haustoria were summed up as shown in a separate diagram in the upper right corner. each category of plant defense responses was tested for significant differences between plants with and without silencing using t-test and this revealed no significant differences. it was confirmed in an independent experiment that cyp96b silencing had no effect on the frequency of haustoria. discussion during recent years, large transcriptome profiling has become a routine analysis in the study of plant responses against abiotic and biotic stresses and was considered as a promising tool on the way to improve crop performance. here, we report on a cytochrome p gene of barley which was identified in a macroarray-based study as differentially regulated during nonhost and host interactions with fungal isolates of the magnaporthe species complex. we provide evidence that this gene is involved in execution of penetration resistance as an early response to the ectoparasitic growth of the pathogen on the leaf surface. starting in the leibniz institute of plant genetics and crop plant research at gatersleben developed a macroarray on the basis of expressed sequence tags and made it available as a resource for the barley community to study gene expression in response to various treatments. using this array, we identified a set of barley genes whose transcripts accumulate to a higher dose or at earlier time points after inoculation with a magnaporthe nonhost as compared to a magnaporthe host isolate. a prominent member among these genes was an est clone identical to the barley mloc_ cdna which has a domain characteristic for cytochrome p enzymes. plant p450s are involved in primary and secondary metabolic processes and catalyze different monooxygenation and hydroxylation reactions. the wide distribution of p450s in a plant species-specific manner implies that they are subject to high evolutionary pressure which led to diversification and finally to novel metabolic pathways and products with acquired new biological functions. search for homology among plant p450s placed the mloc_ protein into the cyp family and suggested an enzymatic function as midchain alkane hydroxylase. the closest arabidopsis homologue of cyp96b is cyp96a, which was shown to be a splicing variant of a monocistronic transcript, forming a dimer with cyp96a9. it was suggested that the dimerization is required for the modification of hydrophobic substrates. in arabidopsis cyp96a transcripts were found less abundant than cyp96a transcripts, accounting for cyp96a concentration as a rate limiting factor at least at the level of transcriptional regulation. assuming a similar situation for barley, this could be interpreted as if cyp96b has gained a novel regulatory function in the resistance response of barley against magnaporthe nonhost isolates. in this scenario, the transcriptional up-regulation of cyp96b will lead to enhanced formation of heterodimers with a putative barley protein homologous to cyp96a and finally to more production of a hydrophobic product. so far the only cyp enzyme characterized in more detail is arabidopsis mah which provides secondary alcohols and ketones as building blocks for the cuticular wax layer. this outermost surface layer is the first barrier which plant pathogens have to cope with during infection. additionally, the cuticle might also be a reservoir for the release of signal molecules which could be perceived by the attacked plant itself as damage-associated molecular patterns leading to the execution of defense responses. alternatively, cuticle constituents could be used by pathogens as host identification factors which was shown e.g. in the barley-powdery mildew interaction for a c26-aldehyde. cuticle components may also function in bacterial pathogenicity, e.g. it was shown that the arabidopsis cyp86a a plant p which is involved in cuticle development, is required for the induction of the bacterial type iii secretion system. on the other hand it was shown that free cutin monomers, most likely released by the activity of fungal derived cutinases, may act as endogenous damps and induce plant defense responses. in arabidopsis the main epicuticular wax components are alkanes, secondary alcohols and ketones with a chain length of c all of which are synthesized via a mah1-dependent pathway. however, barley cuticles mainly contain a c26-primary alcohol, n-hexacosanol, whose synthesis does not require a mah enzymatic conversion. it might be, therefore, that mah1-derived wax components in barley are less important against pathogen attack as structural barriers but instead act in signaling. we addressed the biological function of cyp96b in a transient assay using bsmv as a tool to knock-down gene expression. the usefulness of bsmv-mediated vigs for functional genomics in wheat and barley has been demonstrated in several recent publications and we already adopted the assay for the study of barley-magnaporthe interactions. since it was shown for wheat that bsmv-infection prior to inoculation with m. oryzae host isolates may lead to decreased penetration, we used in all of our vigs-experiments plants inoculated with bsmv without silencing construct as an internal control. doing so and comparing these control plants with those showing the highest reduction in cyp96b transcript abundance, a significant decrease in the frequency of papillae at sites of attempted penetration by the magnaporthe nonhost isolate cd was observed. since this latter result went along with more epidermal cells showing deposition of autofluorescent material at entire cell walls, this is indicative of ineffective papillae which could not block magnaporthe penetration. the more rapid transition of fungal invasion across cell walls became even more obvious in the barley host interaction with m. oryzae isolate th where cyp96b silencing led to an increase of invasive hyphae in attacked epidermal cells. similar findings for decreased penetration defense and more frequent entry of pathogens into epidermal cells, were also reported for arabidopsis pen-mutants. in the light of this finding cyp96b could be regarded as being a barley pen-gene. however, neither in arabidopsis pen-mutants nor in our experiments with cyp96b22-vigs, nonhost pathogens were able to sporulate, indicating effective post-penetration resistance mechanisms. disabling this second line of defense by blocking the eds1-pad4-sag signaling complex renders arabidopsis fully susceptible even to non-adapted pathogens. in barley, the deposition of autofluorescent material seems to be a cytological marker for this back-up strategy in interactions with magnaporthe. nonhost resistance is generally considered to be determined by several quantitative trait loci all of which must act in common to prevent infection by a would-be pathogen. only the detailed monitoring of minor cytological changes in bsmv-vigs treated barley plants enabled us to assign a function in penetration resistance against magnaporthe nonhost isolates to the barley gene cyp96b it would be advisable, therefore, to refrain from macroscopic evaluation in nonhost analysis after knock-out or knock-down of candidate gene expression. since the infection process of the adapted powdery mildew fungus on barley plants after knock-down of cyp96b expression was not altered, we could rule out the possibility of an entire collapse of penetration resistance in these plants. the finding that cyp96b may act differently in barley powdery mildew vs. magnaporthe interactions was not astonishing, since similar observations had been made before for barley mlo or rop genes. this ambivalence in gene regulation was interpreted as part of a dedicated response of barley to biotrophic or hemi-biotrophic pathogens. interestingly, cyp96b was found to be down-regulated in barley after inoculation with puccinia triticina and puccinia hordei. however a functional verification of this result e.g. in a bsmv-vigs assay has not been reported yet. an unexpected finding of the present study was the sensing of the ectoparasitic growth of magnaporthe germ tubes at the leaf surface by barley plants. using the m. oryzae mutant Δpmk we undoubtedly verified that penetration is not necessary to trigger cyp96b expression. we showed also for magnaporthe nonhost isolates, for which no Δpmk mutants existed, that they were able to cause the same phenotype by using a fungicide leading to non-functional appressoria and thereby preventing penetration. other studies also indicated a signal exchange between pathogens and plants before penetration, however the chemical nature of the signal itself was never elucidated. most recently it was shown for arabidopsis that effectors of colletotrichum higginsianum are secreted before penetration. the homology of cyp96b to mah enzymes and its implication in synthesis of cuticular waxes leads us to speculate that mah1-dependent waxy components might be deliberated during the mucilage-facilitated attachment of spores and germ tubes to the leaf surface. thereafter they are sensed as damps and lead to increased defense responses associated with penetration resistance as e.g. the formation of effective papilla. in the host situation, m. oryzae isolates might then secrete effector molecules which negate these defense reactions.  our results provide functional evidence that the barley cytochrome p gene cyp96b plays a crucial role in execution of the nonhost response of barley against pathogens from the magnaporthe species complex. in the light of head blast as becoming a more prevalent disease in barley and wheat cultivation, our study might provide useful information for further breeding strategies. however, more studies are needed to establish whether the deduced protein functions in strengthening of structural barriers against pathogen attack or in generation of signal molecules. 
we propose a functional overlap of cyp96b in the execution of penetration resistance during basal and nonhost resistance of barley against different magnaporthe species. taken together our results suggest a cross-talk between barley and magnaporthe isolates across the plant surface. usage of a fungicidal treatment and a magnaporthe mutant confirmed that penetration was not necessary for this early activation of cyp96b transcriptional silencing of cyp96b using barley stripe mosaic virus led to a decrease in penetration resistance of barley plants to magnaporthe host and nonhost isolates. array experiments identified a barley gene encoding a putative cytochrome p monooxygenase whose transcripts accumulate to a higher concentration in the nonhost as compared to the host interaction. head blast caused by the fungal plant pathogen magnaporthe oryzae is an upcoming threat for wheat and barley cultivation. as a potential source for novel resistance traits. we investigated the nonhost response of barley to an isolate of the magnaporthe species complex which is pathogenic on pennisetum spp. since members of the plant cyp family are known to be involved in synthesis of epicuticular waxes, these substances or their derivatives might act as signal components. the gene clusters within the cyp clade of the p plant gene family and is designated as cyp96b expression of cyp96b was triggered during the ectoparasitic growth of the pathogen on the outside of the leaf.
 head blast caused by the fungal plant pathogen magnaporthe oryzae is an upcoming threat for wheat and barley cultivation. we investigated the nonhost response of barley to an isolate of the magnaporthe species complex which is pathogenic on pennisetum spp. as a potential source for novel resistance traits.  array experiments identified a barley gene encoding a putative cytochrome p monooxygenase whose transcripts accumulate to a higher concentration in the nonhost as compared to the host interaction. the gene clusters within the cyp clade of the p plant gene family and is designated as cyp96b expression of cyp96b was triggered during the ectoparasitic growth of the pathogen on the outside of the leaf. usage of a fungicidal treatment and a magnaporthe mutant confirmed that penetration was not necessary for this early activation of cyp96b transcriptional silencing of cyp96b using barley stripe mosaic virus led to a decrease in penetration resistance of barley plants to magnaporthe host and nonhost isolates. this phenotype seems to be specific for the barley-magnaporthe interaction, since penetration of the adapted barley powdery mildew fungus was not altered in similarly treated plants.  taken together our results suggest a cross-talk between barley and magnaporthe isolates across the plant surface. since members of the plant cyp family are known to be involved in synthesis of epicuticular waxes, these substances or their derivatives might act as signal components. we propose a functional overlap of cyp96b in the execution of penetration resistance during basal and nonhost resistance of barley against different magnaporthe species. nonhost resistancemagnaporthe oryzaehead blastcytochrome p450waxcuticlepenetrationbsmv-vigs 