This large number of intergenic transcripts suggests that noncoding RNA may play a significant role in transcriptional regulation. The results also indicate that almost 50% more rRNA transcripts are generated at the lower temperature consistent with high levels of aflatoxin production. Among the 13 487 known genes signaling pathway in the A. flavus genome, 72% were expressed under both conditions. Overall, 8626 genes were not significantly affected by the growth temperature, while 1153 were

differentially expressed. Among the latter, 551 genes had higher expression levels, while 602 genes had lower expression levels at lower temperature. Notably, six times more genes were highly upexpressed at 30 °C. Thus, 77 genes were highly upexpressed, while only 12 were highly downexpressed at that temperature. Most of the highly upexpressed genes were involved LGK-974 nmr in aflatoxin biosynthesis as discussed below. To evaluate the effect of temperature on the regulation of secondary metabolite biosynthesis, we used the smurf program (http://www.jcvi.org/smurf) (Khaldi et al., 2010) to identify putative secondary metabolite gene clusters (Table S2). Among the 55 clusters identified in the A. flavus genome, 11 clusters were upregulated (clusters #1, 11, 13, 23, 20, 21, 30, 43, 45, 54 and 55), while only two clusters were downregulated (cluster #2 and 3) at lower temperature.

Among upregulated clusters three were associated with known products: conidial pigment (cluster #10), aflatoxin (cluster #54) and cyclopiazonic acid (CPA) (cluster #55). Further analysis of the aflatoxin biosynthesis cluster quantitatively demonstrated that aflatoxin production is one of the most tightly regulated processes in a fungal cell. Most genes in C59 the aflatoxin cluster were highly upexpressed at 30 °C, while not expressed at 37 °C (Table 1). The five most highly expressed genes encoded the following enzymes:

reductase AflD, ketoreductase AflM, alcohol dehydrogenase AflH, O-methyltransferase AflO and VERB synthase AflK. Notably, adjacent sugar utilization genes (nadA, hxtA, glcA and sugR) (Yu et al., 2000), had higher expression levels under conditions nonconducive to aflatoxin production. This suggests that they are not controlled by the aflatoxin pathway regulatory genes and not directly involved in aflatoxin biosynthesis contrary to previous reports (Yu et al., 2000, 2004a, b). Intriguingly, aflR and aflS (formerly designated aflJ), the two transcriptional regulators of the aflatoxin biosynthesis pathway, were expressed at both temperature conditions. Their expression levels were five and 24 times higher, respectively, at the lower temperature. They were among the three most expressed genes in the cluster at the higher temperature. It was hypothesized previously that AflS binds to AflR to prevent inhibitor binding and to allow for the aflatoxin pathway transcription (Chang, 2004).