Effects of Aspergillus flavus colonization of maize kernels under different water activities (a; 0.99 and 0.91) and temperatures (30, 37°C) on (a) aflatoxin B (AFB) production and (b) the transcriptome using RNAseq were examined. There was no significant difference (p=0.05) in AFB production at 30 and 37°C and 0.99 a. However, there was a significant (p=0.05) increase in AFB at 0.91 a at 37°C when compared with 30°C/0.99 a. Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 a showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30°C and 702 genes at 37°C. Increasing temperature from 30 to 37°C at both a levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing a at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both a and temperature. An interaction effect for 4 of the 25 AFB genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by a stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed.

Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28s rRNA loop required for elongation factor l u binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulator may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants. INTRODUCTIONRibosome-inactivating proteins (RIPs) are a widely distributed group of toxic plant proteins that catalytically inactivate eukaryotic ribosomes (for review, see Stirpe and Barbieri, 1986). RlPs function as N-glycosidases to remove a specific adenine in a conserved loop of the large rRNA . This irreversible modification renders the ribosome unable to bind elongation factor l a , thereby blocking translation. Because this translational inhibitory activity is toxic, RlPs have been tested extensively for use as immunotoxins and antiviral agents and more recently for their effects on protozoa, insects, and fungi (Barbieri and Stirpe, 1982;Cenini et al., 1988;Gatehouse et al., 1990;Leah et al., 1991).RIP activities have been found in the seed, root, leaf, or sap of more than 50 different plant species (Gasperi-Campani et al., 1985). Two forms of RlPs have been described (Stirpe and Barbieri, 1986). Type 1 RlPs such as pokeweed antiviral protein, trichosanthin, the barley translation inhibitor, and gelonin are monomeric enzymes, each with an approximate M, of 30,000 (Irvin, 1975;Stirpe et al., 1980;Asano et al., 1984;Maraganore et al., 1987;Yeung et al., 1988). Type 2 RlPs such as ricin, abrin, and modeccin are highly toxic heterodimeric proteins, each with an approximate M, of 60,000 in which one polypeptide with RIP activity (A-chain) is linked by a disulfide bridge to a galactose-binding lectin (B-chain; Pihl, 1973, 1982;Stirpe et al., 1978).Type 1 RlPs and the A-chain of type 2 RlPs have basic isoelectric points, and many have signal peptides that are not To whom correspondence should be addressed. present in the mature protein (Stirpe and Barbieri, 1986). Although RlPs share biological activity, they typically exhibit similarities of <50%, and antibodies raised against RlPs seldom cross-react with RlPs from distantly related species (Ready et al., 1988). The maize b-32 protein has homology with severa1 previously characterized RIPs, yet it is a singlechain acidic protein tha...

Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understanding host response to infection by the fungus, transcription of approximately 9000 maize genes were monitored during the host-pathogen interaction with a custom designed Affymetrix GeneChip® DNA array. More than 4000 maize genes were found differentially expressed at a FDR of 0.05. This included the up regulation of defense related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel by A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.

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.