Research into late embryogenesis abundant (LEA) proteins has been ongoing for more than 20 years but, although there is a strong association of LEA proteins with abiotic stress tolerance particularly dehydration and cold stress, for most of that time, their function has been entirely obscure. After their initial discovery in plant seeds, three major groups (numbered 1, 2 and 3) of LEA proteins have been described in a range of different plants and plant tissues. Homologues of groups 1 and 3 proteins have also been found in bacteria and in certain invertebrates. In this review, we present some new data, survey the biochemistry, biophysics and bioinformatics of the LEA proteins and highlight several possible functions. These include roles as antioxidants and as membrane and protein stabilisers during water stress, either by direct interaction or by acting as molecular shields. Along with other hydrophilic proteins and compatible solutes, LEA proteins might also serve as "space fillers" to prevent cellular collapse at low water activities. This multifunctional capacity of the LEA proteins is probably attributable in part to their structural plasticity, as they are largely lacking in secondary structure in the fully hydrated state, but can become more folded during water stress and/or through association with membrane surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the various in vitro defined functions in the living cell: Are the LEA proteins truly multi-talented, or are they still just misunderstood?

The continuing conundrum of the LEA proteins

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

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Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for "hot topic" research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

Trichoderma reesei (Hypocrea jecorina) is an efficient cell factory for protein production that is exploited by the enzyme industry. Yields of over 100 g secreted protein l−1 from industrial fermentations have been reported. In this review we discuss the spectrum of proteins secreted by T. reesei and the studies carried out on its protein secretion system. The major enzymes secreted by T. reesei under production conditions are those degrading plant polysaccharides, the most dominant ones being the major cellulases, as demonstrated by the 2D gel analysis of the secretome. According to genome analysis, T. reesei has fewer genes encoding enzymes involved in plant biomass degradation compared with other fungi with sequenced genomes. We also discuss other T. reesei secreted enzymes and proteins that have been studied, such as proteases, laccase, tyrosinase and hydrophobins. Investigation of the T. reesei secretion pathway has included molecular characterization of the pathway components functioning at different stages of the secretion process as well as analysis of the stress responses caused by impaired folding or trafficking in the pathway or by expression of heterologous proteins. Studies on the transcriptional regulation of the secretory pathway have revealed similarities, but also interesting differences, with other organisms, such as a different induction mechanism of the unfolded protein response and the repression of genes encoding secreted proteins under secretion stress conditions.

The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina)

Trichoderma reesei was cultivated in chemostat cultures on lactose-containing medium. The cultures were characterized for growth, consumption of the carbon source and protein production. Secreted proteins were produced most efficiently at low specific growth rates, 0.022-0.033 h(-1), the highest specific rate of total protein production being 4.1 mg g(-1) h(-1) at the specific growth rate 0.031 h(-1). At low specific growth rates, up to 29 % of the proteins produced were extracellular, in comparison to only 6-8 % at high specific growth rates, 0.045-0.066 h(-1). To analyse protein synthesis and secretion in more detail, metabolic labelling of proteins was applied to analyse production of the major secreted protein, cellobiohydrolase I (CBHI, Cel7A). Intracellular and extracellular labelled CBHI was quantified and analysed for pI isoforms in two-dimensional gels, and the synthesis and secretion rates of the molecule were determined. Both the specific rates of CBHI synthesis and secretion were highest at low specific growth rates, the optimum being at 0.031 h(-1). However, at low specific growth rates the secretion rate/synthesis rate ratio was significantly lower than that at high specific growth rates, indicating that at low growth rates the capacity of cells to transport the protein becomes limiting. In accordance with the high level of protein production and limitation in the secretory capacity, the transcript levels of the unfolded protein response (UPR) target genes pdi1 and bip1 as well as the gene encoding the UPR transcription factor hac1 were induced.

The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei.

Proteins destined to be secreted from eukaryotic cells pass through secretory organelles, namely, the endoplasmic reticulum (ER) and the Golgi complex, and are finally transported to the plasma membrane to be released from the cell. The proteins are transported between the organelles and to the plasma membrane in carrier vesicles. Each vesicle transport step includes packaging of the cargo proteins and budding of the vesicle from the donor compartment membrane, migration of the vesicle to the acceptor compartment, and docking and fusion of the vesicle to the acceptor compartment membrane.

In order to maintain cellular organization, each vesicle fusion must be specific for the right acceptor membrane. This specificity is ensured by protein factors that are specific only for a given fusion event. The specificity of fusion is largely due to recognition proteins in the vesicle and target membranes (v-SNAREs and t-SNAREs, respectively), but other types of stage-specific proteins that assist in vesicle docking and fusion are also important. The small GTP-binding Rab proteins belonging to the Ras superfamily associate with the vesicle membrane through lipid modification in their GTP-bound active form (19). They have a role in the formation of a large protein complex (20S complex; includes the t-SNARE and v-SNARE) that is needed for membrane fusion to take place (43). It has been discovered that during the assembly of this complex, the Saccharomyces cerevisiae Rab protein involved in ER-Golgi transport, Ypt1p, interacts transiently with the corresponding t-SNARE, Sed5p, displacing the negative regulator Sly1p and allowing interaction of the t-SNARE and v-SNARE (21, 37). According to recent evidence, Ypt1p is also involved in protein sorting upon exit from the ER (26).

In addition to proteins specific for one fusion step, general fusion factors that are functional for multiple membrane fusion steps are also needed in the secretory pathway. These include yeast Sec18p and its mammalian counterpart, NSF (11, 55, 56). Sec18p has been isolated as a member of the membrane fusion protein complex (20S complex) (53). It was believed that hydrolysis of ATP by Sec18p/NSF triggers dissociation of the fusion complex and subsequently fusion of the vesicle and target membranes (6, 44). More recent papers, however, suggest that mammalian NSF activates the SNARE proteins prior to membrane fusion (29) or that it participates in their recycling after membrane fusion (52), perhaps by acting as a chaperone that alters the conformation of the SNAREs (27, 30).

Filamentous fungi are naturally good protein secretors, and because of the high efficiency of secretion by industrially exploited fungi such as Trichoderma reesei and Aspergillus niger, the secretory pathways of these fungi are of particular interest. Knowledge at the molecular level about intracellular protein folding and trafficking in filamentous fungi is beginning to accumulate (reviewed in reference 7). Folding factors, such as Bip and protein disulfide isomerase (PDI), have been characterized at the gene level for some filamentous fungi (e.g., see references 28, 38, and 48). Of the genes involved in protein trafficking, results have hitherto been published for the ypt1 genes of Neurospora crassa (13) and Phytophthora infestans (5) and for homologs of the yeast SAR1 gene from A. niger and T. reesei (49). Punt et al. (36) have cloned several secretion-related small GTPases from A. niger, and Dumas et al. (8) have cloned a homolog of yeast SEC4 from Colletotrichum lindemuthianum. In this paper, we report the characterization of the homologs of the yeast YPT1 and SEC18 genes from the filamentous fungus T. reesei and, for comparison, from A. niger var. awamori. The regulation of the T. reesei genes in the presence of inhibitors of protein secretion was studied, and evidence is presented to explain the transcriptional regulation of the Trichoderma secretory pathway.

Characterization of Secretory Genes ypt1/yptA and nsf1/nsfA from Two Filamentous Fungi: Induction of Secretory Pathway Genes of Trichoderma reesei under Secretion Stress Conditions

We have isolated the birch homologue (BP8) for the carrot embryogenic gene DC8 by heterologous hybridization. The birch BP8 gene encodes a putative protein of 53 kDa, showing 52% sequence identity with the DC8 gene at the amino acid level. The putative BP8 protein contains 20 repeats of 11 amino acids and thus belongs to the group of LEA proteins isolated from such plants as carrot, cotton and wheat. Northern hybridization of mRNA isolated from birch cells representing different stages of somatic embryogenesis and non-embryogenetic material with a PB8 probe gave no signals, suggesting a low expression level of the BP8 gene.

Characterization of a birch (Betula pendula Roth.) embryogenic gene, BP8.

Characterisation of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components.

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38°C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2001.02716.x

Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei.

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant (rho3Gly20Val alleles of the T. reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

Tuija Vasara

Papers

Characterization of Secretory Genes ypt1/yptA and nsf1/nsfA from Two Filamentous Fungi: Induction of Secretory Pathway Genes of Trichoderma reesei under Secretion Stress Conditions

Characterization of a birch (Betula pendula Roth.) embryogenic gene, BP8.

Characterisation of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components.

Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei.

Trichoderma reesei rho3 a homologue of yeast RH03 suppresses the growth defect of yeast sec15-1 mutation.