A maize lectin-like protein with antifungal activity against Aspergillus flavus.

The filamentous fungus Aspergillus flavus causes an ear rot on maize and produces a mycotoxin (aflatoxin) in colonized maize kernels. Aflatoxins are carcinogenic to humans and animals upon ingestion. Aflatoxin contamination results in a large loss of profits and marketable yields for farmers each year. Several research groups have worked to pinpoint sources of resistance to A. flavus and the resulting aflatoxin contamination in maize. Some maize genotypes exhibit greater resistance than others. A proteomics approach has recently been used to identify endogenous maize proteins that may be associated with resistance to the fungus. Research has been conducted on cloning, expression, and partial characterization of one such protein, which has a sequence similar to that of cold-regulated proteins. The expressed protein, ZmCORp, exhibited lectin-like hemagglutination activity against fungal conidia and sheep erythrocytes. Quantitative real-time PCR assays revealed that ZmCOR is expressed 50% more in maize kernels from the Mp420 line, a type of maize resistant to A. flavus, compared with the expression level of the gene in the susceptible B73 line. ZmCORp exhibited fungistatic activity when conidia from A. flavus were exposed to the protein at a final concentration of 18 mM. ZmCORp inhibited the germination of conidia by 80%. A 50% decrease in mycelial growth resulted when germinated conidia were incubated with the protein. The partial characterization of ZmCORp suggests that this protein may play an important role in enhancing kernel resistance to A. flavus infection and aflatoxin accumulation.

A maize trypsin inhibitor (ZmTIp) with limited activity against Aspergillus flavus.

Infection of maize both pre- and postharvest by Aspergillus flavus is a severe agricultural problem in the southern United States. Aflatoxins are secondary metabolites produced by A. flavus and are carcinogenic to humans and animals upon ingestion. Extensive research has been conducted to identify sources of resistance to A. flavus in maize. Some maize genotypes exhibit greater resistance to A. flavus than others. Many research groups have validated the role of plant trypsin inhibitors (TIs) as a means of plant defense against fungal infection. Research consisting of the cloning, expression, and partial characterization of one previously uncharacterized TI protein has been conducted. The overexpressed protein displayed TI activity, as expected, and some ability to alter germination of conidia (8%) from several fungal pathogens and to inhibit hyphal growth (30%). This effect on fungal growth, although less than that of previously investigated TIs, marks this protein as a potential source of resistance to aflatoxin accumulation in maize.

Identification of Maize Kernel Endosperm Proteins Associated with Resistance to Aflatoxin Contamination by Aspergillus flavus.

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize (Zea mays). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics, and resistance-associated proteins were identified. Here, we report the comparison of maize endosperm proteins from five resistant and five susceptible genotypes, and the identification of additional resistance-associated proteins using the same approach. Ten protein spots were upregulated twofold or higher in resistant lines compared with susceptible ones. Peptide sequencing of these proteins identified them as a globulin-2 protein, late embryogenesis abundant proteins (LEA3 and LEA14), a stress-related peroxiredoxin antioxidant (PER1), heat-shock proteins (HSP17.2), a cold-regulated protein (COR), and an antifungal trypsin-inhibitor protein (TI). The gene encoding one such upregulated protein, PER1, was cloned and overexpressed in Escherichia coli. The overexpressed PER1 protein demonstrated peroxidase activity in vitro. In addition, per1 expression was significantly higher in the resistant genotype Mp420 than in the susceptible genotype B73 during the late stage of kernel development, and was significantly induced upon A. flavus infection, suggesting that it may play an important role in enhancing kernel stress tolerance and aflatoxin resistance. The significance of other identified proteins to host resistance and stress tolerance also is discussed.

What can comparative genomics tell us about species concepts in the genus Aspergillus?

Understanding the nature of species" boundaries is a fundamental question in evolutionary biology. The availability of genomes from several species of the genus Aspergillus allows us for the first time to examine the demarcation of fungal species at the whole-genome level. Here, we examine four case studies, two of which involve intraspecific comparisons, whereas the other two deal with interspecific genomic comparisons between closely related species. These four comparisons reveal significant variation in the nature of species boundaries across Aspergillus. For example, comparisons between A. fumigatus and Neosartorya fischeri (the teleomorph of A. fischerianus) and between A. oryzae and A. flavus suggest that measures of sequence similarity and species-specific genes are significantly higher for the A. fumigatus - N. fischeri pair. Importantly, the values obtained from the comparison between A. oryzae and A. flavus are remarkably similar to those obtained from an intra-specific comparison of A. fumigatus strains, giving support to the proposal that A. oryzae represents a distinct ecotype of A. flavus and not a distinct species. We argue that genomic data can aid Aspergillus taxonomy by serving as a source of novel and unprecedented amounts of comparative data, as a resource for the development of additional diagnostic tools, and finally as a knowledge database about the biological differences between strains and species.

The effect of elevated temperature on gene transcription and aflatoxin biosynthesis.

The molecular regulation of aflatoxin biosynthesis is complex and influenced by several environmental conditions; one of these is temperature. Aflatoxins are produced optimally at 28-30 C, and production decreases as temperatures approach 37 C, the optimum temperature for fungal growth. To better characterize the influence of temperature on aflatoxin biosynthesis, we monitored the accumulation of aflatoxin and the expression of more than 5000 genes in Aspergillus flavus at 28 C and 37 C. A total of 144 genes were expressed differentially (P < 0.001) between the two temperatures. Among the 103 genes more highly expressed at 28 C, approximately 25% were involved in secondary metabolism and about 30% were classified as hypothetical. Genes encoding a catalase and superoxide dismutase were among those more highly expressed at 37 C. As anticipated we also found that all the aflatoxin biosynthetic genes were much more highly expressed at 28 C relative to 37 C. To our surprise expression of the pathway regulatory genes aflR and aflS, as well as aflR antisense, did not differ between the two temperatures. These data indicate that the failure of A. flavus to produce aflatoxin at 37 C is not due to lack of transcription of aflR or aflS. One explanation is that AFLR is nonfunctional at high temperatures. Regardless, the factor(s) sensing the elevated temperatures must be acute. When aflatoxin-producing cultures are transferred to 37 C they immediately stop producing aflatoxin.

Whole genome comparison of Aspergillus flavus and A. oryzae.

Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is Generally Regarded As Safe (GRAS). Whole genome sequences for these two fungi are now complete, providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. These two fungi are very similar in genome size and number of predicted genes. The estimated genome size (36·8 Mb) and predicted number of genes (12 197) for A. flavus is similar to that of A. oryzae (36·7 Mb and 12 079, respectively). These two fungi have significantly larger genomes than Aspergillus nidulans (30·1) and Aspergillus fumigatus (29·4). The A. flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in the predicted number of polyketide synthases, nonribosomal peptide synthases or the number of genes coding for cytochrome P450 enzymes. A micro-scale analysis of the two fungi did show differences in DNA correspondence between the two species and in the number of transposable elements. Each species has approximately 350 unique genes. The high degree of sequence similarity between the two fungi suggests that they may be ecotypes of the same species and that A. oryzae has resulted from the domestication of A. flavus.

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production.

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus.

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.

Characterization of the Aspergillus parasiticus major nitrogen regulatory gene, areA.

The major nitrogen regulatory gene, areA, was cloned from Aspergillus parasiticus. It encoded a polypeptide of 864 amino acids which contained a nuclear localization signal (NLS), a highly acidic region from positions 497 to 542, a Cys-X(2)-Cys-X(17)-Cys-X(2)-Cys DNA-binding motif and a conserved carboxy-terminus. Electrophoretic mobility shift assays suggested that the A. parasiticus AREA DNA-binding domain fusion protein bound cooperatively to single GATA elements in the A. parasiticus niaD-niiA intergenic region. AREA also bound to the aflR-aflJ intergenic region of the aflatoxin biosynthesis gene cluster. Regions of areA were fused to a yeast GAL4 DNA-binding domain coding region to localize putative transcription activation domain(s) of AREA based on activation of the GAL1(p)::lacZ reporter gene expression. The portion between NLS and the acidic domain demonstrated 16-20-fold higher activation activities than other portions of AREA, which suggests that the transcription activation domain is located in this region.

Phytochemical glyceollins, isolated from soy, mediate antihormonal effects through estrogen receptor alpha and beta.

The flavonoid family of phytochemicals, particularly those derived from soy, has received attention regarding their estrogenic activity as well as their effects on human health and disease. In addition to these flavonoids other phytochemicals, including phytostilbene, enterolactone, and lignans, possess endocrine activity. The types and amounts of these compounds in soy and other plants are controlled by both constitutive expression and stress-induced biosynthesis. The health benefits of soy-based foods may, therefore, be dependent upon the amounts of the various hormonally active phytochemicals within these foods. The aim was to identify unique soy phytochemicals that had not been previously assessed for estrogenic or antiestrogenic activity. Here we describe increased biosynthesis of the isoflavonoid phytoalexin compounds, glyceollins, in soy plants grown under stressed conditions. In contrast to the observed estrogenic effects of coumestrol, daidzein, and genistein, we observed a marked antiestrogenic effect of glyceollins on ER signaling, which correlated with a comparable suppression of 17 beta-estradiol-induced proliferation in MCF-7 cells. Further evaluation revealed greater antagonism toward ER alpha than ER beta in transiently transfected HEK 293 cells. Competition binding assays revealed a greater affinity of glyceollins for ER alpha vs. ER beta, which correlated to greater suppression of ER alpha signaling with higher concentrations of glyceollins. In conclusion, we describe the phytoalexin compounds known as glyceollins, which exhibit unique antagonistic effects on ER in both HEK 293 and MCF-7 cells. The glyceollins as well as other phytoalexin compounds may represent an important component of the health effects of soy-based foods.

Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species.

The biosynthesis of aflatoxins (B(1), G(1), B(2), and G(2)) is a multi-enzyme process controlled genetically by over 20 genes. In this study, we report the identification and characterization of the avfA gene, which was found to be involved in the conversion of averufin (AVF) to versiconal hemiacetal acetate (VHA), in Aspergillus parasiticus and A. flavus; a copy of avfA gene was also cloned from a non-aflatoxin producing strain A. sojae. Complementation of an averufin-accumulating, non-aflatoxigenic mutant strain of A. parasiticus, SRRC 165, with the avfA gene cloned from A. flavus, restored the ability of the mutant to convert AVF to VHA and to produce aflatoxins B(1), G(1), B(2), and G(2). Sequence analysis revealed that a single amino acid replacement from aspartic acid to asparagine disabled the function of the enzyme in the mutant strain SRRC 165. The A. parasiticus avfA was identified to be a homolog of previously sequenced, but functionally unassigned transcript, stcO, in A. nidulans based on sequence homology at both nucleotide (57%) and amino acid (55%) levels. In addition to avfA, another aflatoxin pathway gene, omtB, encoding for an O-methyltransferase involved in the conversion of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST), was cloned from A. parasiticus, A. flavus, and A. sojae. The omtB gene was found to be highly homologous to stcP from A. nidulans, which has been reported earlier to be involved in a similar enzymatic step for the sterigmatocystin formation in that species. RT-PCR data demonstrated that both the avfA and avfA1 as well as omtB genes in A. parasiticus were expressed only in the aflatoxin-conducive medium. An analysis of the degrees of homology for the two reported genes between the Aspergillus species A. parasiticus, A. flavus, A. nidulans and A. sojae was conducted.

Cloning and characterization of a novel polygalacturonase-encoding gene from Aspergillus parasiticus.

Pectinases produced by Aspergillus flavus and A. parasiticus are believed to play a significant role in the ability of these fungi to spread in cotton bolls and other crops. Utilizing a DNA probe, generated by PCR, of the Aspergillus niger pgaII gene, we have isolated a novel, constitutively expressed polygalacturonase (PG)-encoding gene (pecA) from an A. parasiticus cDNA library. DNA sequence analysis and the deduced amino acid (aa) sequence of pecA demonstrated significant identity at the nucleotide and aa levels with other PG of fungal origin. Northern blot analysis of RNA isolated from A. parasiticus grown on either glucose or pectin as the sole carbon source showed that pecA was expressed during growth in both media.

Comparison of the omtA genes encoding O-methyltransferases involved in aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus.

O-methyltransferase (OMT) is one of the key enzymes in aflatoxin (AF) biosynthesis in the fungi, Aspergillus flavus (Af) and A. parasiticus (Ap). Genomic DNA clones containing the omtA genes from Ap strain SRRC 143 and Af strain CRA01-2B were sequenced. Comparison of the genomic DNA sequences with the cDNA of this Ap gene revealed the presence of four introns ranging from 52 to 60 bp in length in both species; the region encoding the putative S-adenosylmethionine-binding motif was located between the third and fourth introns. The coding sequence of omtA from Ap strain SRRC 143 demonstrated a greater than 97% sequence identity with that from Af strain CRA01-2B, within the coding region.

Fate of the methyl group during the conversion of sterigmatocystin into O-methylsterigmatocystin and aflatoxin B1 by cell-free preparations of Aspergillus parasiticus.

Cell-free extracts of fungal mycelia of two aflatoxin non-producing isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were utilized for the study of enzyme activities involved in the latter stages of aflatoxin biosynthesis. The post-microsomal fractions (105,000 x g supernatant) of both SRRC 163 and SRRC 2043 were able to convert sterigmatocystin (ST) into O-methylsterigmatocystin (OMST); whereas the microsomal (105,000 x g pellet) preparation of only SRRC 163 was able to convert OMST into aflatoxin B1 (AFB1). S-Adenosylmethionine (SAM) was the primary substrate for the ST to OMST (methyltransferase) enzymatic conversion; [3H]OMST of specific activity 0.93 Ci/mmol was obtained in a reaction containing the [3H]SAM substrate (specific activity 1 Ci/mmol). After the terminal enzymatic conversion of OMST into AFB1, none of the radiolabel of the methyl group from OMST was found in AFB1. It is postulated that the methylation of ST may be required for subsequent enzymatic oxidation of OMST to aflatoxin B1.

Effects of volatile aldehydes from Aspergillus-resistant varieties of corn on Aspergillus parasiticus growth and aflatoxin biosynthesis.

The fungi Aspergillus flavus and Aspergillus parasiticus produce a potent class of hepatocarcinogens known as aflatoxins. Corn-derived volatile compounds have been previously found to affect growth and aflatoxin production in A. flavus. In this study, the effects on A. parasiticus of three corn-derived volatile compounds, n-decyl aldehyde, hexanal and octanal, were measured. These three compounds were previously found to be variably expressed in five Aspergillus-resistant maize strains and three susceptible strains. In this study, A. parasiticus radial growth was restricted least by n-decyl aldehyde and most by octanal. Treatments of 100 microl of both hexanal and octanal were found to completely inhibit radial growth of the fungus using an agar plate assay method. While the volatile compound n-decyl aldehyde had less of an effect on radial growth than the other volatiles, the n-decyl aldehyde treated colonies had a predominance of uniquely aerial hyphae. These colony structures were found to have more complex hyphae and significantly fewer conidiophores than the control and other aldehyde treatments. Furthermore, aflatoxin production by the fungus was reduced by n-decyl aldehyde and hexanal, but was stimulated by octanal. The results presented here indicate that all three volatile compounds reduce radial growth but only n-decyl aldehyde significantly inhibits aflatoxin biosynthesis in A. parasiticus.

The effects of selected cotton-leaf volatiles on growth, development and aflatoxin production of Aspergillus parasiticus.

The fungi Aspergillus flavus and Aspergillus parasiticus produce the hepatocarcinogenic, secondary metabolites, aflatoxins, in cottonseed, corn, peanuts and treenuts. Results have shown that aflatoxigenic strains of A. flavus and A. parasiticus grown in the presence of specific cotton-leaf volatiles exhibit alterations in aflatoxin production accompanied by variations in growth of the fungi. In this study, two alcohols (3-methyl-1-butanol (3-MB) and nonanol) and two terpenes (camphene and limonene) were chosen as representative cotton-leaf volatiles based on the effects they had on fungal growth and/ or aflatoxin production in previous investigations. The morphological effects of volatile exposure were examined in correlation with fungal growth and aflatoxin production. 3-MB-treated samples exhibited a decrease in fungal radial growth which was directly proportional to the volatile dosage. Additionally, 3-MB treatment resulted in loss of mycelial pigmentation and a decrease in sporulation. Limonene and camphene-treated samples yielded negligible differences in radial growth and morphology when compared to unexposed controls. In addition to radial growth inhibition, samples grown in the presence of nonanol demonstrated uniquely aerial hyphae. In comparison to an unexposed control, aflatoxin production increased in cultures exposed to 3-MB but decreased when exposed to the other three volatiles studied.

Inducers of Aflatoxin Biosynthesis from Colonized Maize Kernels Are Generated by an Amylase Activity from Aspergillus flavus.

ABSTRACT Aflatoxin biosynthesis was induced by compounds in filtrates (EF) obtained from cultures consisting of ground maize kernels colonized by Aspergillus flavus. The inducing activity increased to a maximum at 4 days of incubation and then decreased. Amylase activity was detected in the EF, suggesting that the inducers are products of starch degradation (glucose, maltose, and maltotriose). Analysis of the enzyme by isoelectric focusing electrophoresis indicated a single alpha-amylase with a pI of 4.3. No maltase or amyloglucosidase was detected in the EF. High-pressure liquid chromatography analysis of the EF indicated the presence of glucose, maltose, and maltotriose in near-equal molar concentrations (about 15 mM). With a beta-glucuronidase (GUS) reporter assay consisting of A. flavus transformed with an aflatoxin gene promoter-GUS reporter gene fusion to monitor induction of aflatoxin biosynthesis, the minimum concentration of glucose, maltose, or maltotriose that induced measurable GUS activity was determined to be 1 mM. These results support the hypothesis that the best inducers of aflatoxin biosynthesis are carbon sources readily metabolized via glycolysis. They also suggest that alpha-amylase produced by A. flavus has a role in the induction of aflatoxin biosynthesis in infected maize kernels.

Identification of unique or elevated levels of kernel proteins in aflatoxin-resistant maize genotypes through proteome analysis.

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize (Zea mays L.). Resistant maize genotypes have been identified, but the incorporation of resistance into commercial lines has been slow due to the lack of selectable markers. Here we report the identification of potential markers in resistant maize lines using a proteomics approach. Kernel embryo proteins from each of two resistant genotypes have been compared with those from a composite of five susceptible genotypes using large format two-dimensional gel electrophoresis. Through these comparisons, both quantitative and qualitative differences have been identified. Protein spots have been sequenced, and based on peptide sequence homology analysis, are categorized as follows: storage proteins (globulin 1 and globulin 2), late embryogenesis abundant (LEA) proteins related to drought or desiccation (LEA3 and LEA14), water- or osmo-stress related proteins (WSI18 and aldose reductase), and heat-stress related proteins (HSP16.9). Aldose reductase activity measured in resistant and susceptible genotypes before and after infection suggests the importance of constitutive levels of this enzyme to resistance. Results of this study point to a correlation between host resistance and stress tolerance. The putative function of each identified protein is discussed.

Identification of a maize kernel stress-related protein and its effect on aflatoxin accumulation.

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize. Through proteomic comparisons of maize kernel embryo proteins of resistant and susceptible genotypes, several protein spots previously were found to be unique or upregulated in resistant embryos. In the present study, one of these protein spots was sequenced and identified as glyoxalase I (GLX-I; EC 4.4.1.5). The full-length cDNA of the glyoxalase I gene (glx-I) was cloned. GLX-I constitutive activity was found to be significantly higher in the resistant maize lines compared with susceptible ones. After kernel infection by A. flavus, GLX-I activity remained lower in susceptible genotypes than in resistant genotypes. However, fungal infection significantly increased methylglyoxal (MG) levels in two of three susceptible genotypes. Further, MG was found to induce aflatoxin production in A. flavus culture at a concentration as low as 5.0 muM. The mode of action of MG may be to stimulate the expression of aflR, an aflatoxin biosynthesis regulatory gene, which was found to be significantly upregulated in the presence of 5 to 20 muM MG. These data suggest that GLX-I may play an important role in controlling MG levels inside kernels, thereby contributing to the lower levels of aflatoxins found in resistant maize genotypes.

A Corn Trypsin Inhibitor with Antifungal Activity Inhibits Aspergillus flavus alpha-Amylase.

ABSTRACT In this study, we found that the inhibition of fungal growth in potato dextrose broth (PDB) medium by the 14-kDa corn trypsin inhibitor (TI) protein, previously found to be associated with host resistance to aflatoxin production and active against various fungi, was relieved when exogenous alpha-amylase was added along with TI. No inhibitory effect of TI on fungal growth was observed when Aspergillus flavus was grown on a medium containing either 5% glucose or 1% gelatin as a carbon source. Further investigation found that TI not only inhibited fungal production of extracellular alpha-amylase when A. flavus was grown in PDB medium containing TI at 100 mug ml(-1) but also reduced the enzymatic activity of A. flavus alpha-amylase by 27%. At a higher concentration, however, TI stimulated the production of alpha-amylase. The effect of TI on the production of amyloglucosidase, another enzyme involved in starch metabolism by the fungus, was quite different. It stimulated the production of this enzyme during the first 10 h at all concentrations studied. These studies suggest that the resistance of certain corn genotypes to A. flavus infection may be partially due to the ability of TI to reduce the production of extracellular fungal alpha-amylase and its activity, thereby limiting the availability of simple sugars for fungal growth. However, further investigation of the relationship between TI levels and fungal alpha-amylase expression in vivo is needed.