A ROLE OF CALCIUM SIGNALING GENES IN HETEROKARYON INCOMPATIBILITY IN NEUROSPORA CRASSA

We have studied the Ca-signaling knockout mutants for their role in mating-type-associated heterokaryon incompatibility in Neurospora crassa. The found results showed on heterokaryons homokaryosis for NCU05225, NCU06366, NCU06650, NCU07075, and ∆NCU07966 Ca-signaling knockout mutants (Neurospora crassa unit number, NCU) displayed heterokaryon het compatibility; however heterokaryons heterokaryosis for NCU05225, NCU063665, NCU06650, NCU07075, and ∆NCU07966 mutants displayed het incompatibility like the wild-type control. In addition to that Two Ca-signaling knockout mutants NCU02283, and NCU09655 were tested for mating-type-associated heterokaryon incompatibility; these results showed, heterokaryons homokaryosis and heterokaryons heterokaryosis for NCU02283, NCU09655 mutants displayed het incompatibility. Cell death and hyphal compartmentation due to mating type associated incompatibility were confirmed by uptake of vital dye Evan’s blue. Thus, these results of NCU05225, NCU06366, NCU06650, NCU07075, and NCU07966 Ca-signaling gene products could play a role in mating-type-associated heterokaryon incompatibility in N. crassa. In this article, we are reporting initially screened Ca-signaling gene deletion mutants of these five acts as recessive suppressors of mating type associated vegetative incompatibility in N. crassa.


Introduction
A cell containing different nuclear types in the same cytoplasm is called heterokaryon.
Heterokaryon incompatibility refers to a condition when two nuclei of different genotypes cannot co-exist together in the same cytoplasm ( Fig.1) (Staben and Yanofsky, 1990;Glass et al., 1988, Debets andGriffiths, 1998;Worrall, 1997, Caten, 1972Debets et al., 1994;Van Diepeningen et al., 1998). In N. crassa, both sexual and vegetative heterokaryon systems are present, sexual recognition is restricted by the mating type locus, whereas vegetative het incompatibility is genetically control by particular loci termed as het (for heterokaryon incompatibility) or vic (vegetative heterokaryon incompatibility) (Glass et al., 1988;Ferreira, et al.,1998). When two fungal individuals of distinct het alleles come together, the consequential heterokaryotic cells are quickly damaged or strictly inhibited in their growth (Glass, 2006 andMarek et al., 2003;Saupe, 2000) (Fig.1). This is analogous to histocompatibility system in invertebrates and major histocompatibility (MHC) in mammals Glass and Kaneko, 2003;. N. crassa has two mating (mat) types mat A and mat a. The coexistence of mat A and mat a during the vegetative phase is lethal; however, coexistence of both mating types is necessary for the sexual development (Saupe, 2000;Garnjobst et al., 1956, Wu, 2001Sarkar et al., 2002). These Two mating (mat) types mat A and mat a, alleles are differ in sequences although they occupy the same loci in different strains, and termed as 'idiomorph' (Glass et al., 1988). The A idiomorph is 5301 base pairs that consists of mat A-1,mat A-2 and mat A-3 genes; in contrast, a idiomorph is 3235 base pair and contains only one ORF, mat a-1. The total number of het loci in N. crassa is 11, five of them the mating locus, het-c, -d, -e, and i, they have been recognized using enforced heterokaryons between nearly isogenic strains.
A non-self-recognition process called heterokaryon incompatibility (het) that operates during the vegetative and sexual phases of the filamentous in N. crassa (Glass et al., 1988;Ferreira et al., 1998;Garnjobst, et al., 1956).

Heterokaryon Incompatibility in Other Filamentous Fungi
The fungal mating types which are tremendously dissimilar from each other, and do not show homology between strains of the opposite sex (as opposed to the allelic relationship in most polymorphic systems). The variance between dissimilar allelic forms of a het gene is generally widespread, but single-amino-acid differences can be sufficient to trigger incompatibility (Newmeyer, 1970;Glass et al., 1988;Deleu, 1993). The mat A-1 gene, encode a polypeptide of 288 amino acids region containing amino acids 90-104 has significant similarity to the MAT α1 polypeptide of Saccharomyces cerevisiae , this is required for the expression of the het incompatibility and sexual functions. The mat a-1 encodes a 382 amino acids residues MAT a-1 polypeptide consisting of an HMG domain, and a separate region from amino acid residues 216-220 that confers vegetative incompatibility (vic) in N. crassa (Staben and Yanofsky, 1990;Philley and Staben, 1994). In Podospora anserine a having two het loci, het-s and het-e are functional similar to the that of het-c and het-6 of N. crassa, and vib-1(vegetative incompatibility block-1) of Aspergillus nidulans; but all of these genes are not homologous to each other (Ferreira et al., 1998;Saupe, 2000;Sarkar et al., 2002). The distinguish mechanism of het incompatibility reconcile by allele of differences at the het-c loci of N. crassa that inhibits phenotypic aspects of het-c vegetative incompatibility Glass and Kaneko, 2003;. The molecular description of het loci and het genes participating in the incompatibility effect has been achieved for two ascomycete's N. crassa and P. anserine (Smith et al., 2000;Dementhon et al., 2003;Glass and Kaneko, 2003;Saupe, 2000).

Biological Significance of Heterokaryon Incompatibility
The biological significance of heterokaryon incompatibility, two different views was explained by subject. First, it has been proposed that heterokaryon incompatibility genes continue living limit nutrient situation, and heterokaryon development between different individuals Glass et al., 2004;. Second, the heterokaryotic cells strength boundary to the horizontal gene transfer of cytoplasmic infectious fundamentals elements such as senescence of plasmids, mycoviruses, transposes (Anagnostakis, 1977(Anagnostakis, , 1983Baidyaroy et al., 2000;Biella et al., 2002;Caten, 1972;Debets et al., 1994;Van Diepeningen et al., 1998;Hartl et al., 1975;Hickey et al., 2002;Glass et al., 2004). On the other hand it has been proposed that mating type associated heterokaryon incompatibility might be maintaining out breeding by preventing the construction of different mating types of heterokaryons between siblings of the identical crosses (Debets and Griffiths, 1998;Worrall, 1997).

Present Understanding of Mating Type-Associated Het Incompatibility in N. Crassa
N. crassa has two mating (mat) types, mat A and mat a, both mating types are essential for sexual development, however, coexistence during the vegetative phase is lethal, and therefore display incompatibility. The importance of mating type associated heterokaryon incompatibility, as anticipated for other het genes, the appearance of mating type allied incompatibility might be chance (Sarkar et al., 2002). The tol gene (tol gene, for tolerant) is a suppressor of mating-type-associated heterokaryon incompatibility in N. crassa (Saupe, 2000). The Ca 2+ -signaling genes regulates numerous processes secretion, sporulation, cytoskeletal organization, circadian rhythm, hyphal tip growth and hyphal branching in N. crassa (Fig. 1). Does Calcium signaling genes impact on mating-typeassociated heterokaryon incompatibility in N. crassa? On this foundation, we designed the experiment by using auxotrophic marker for testing for complementation answer on Ca 2+ -signaling genes in N. crassa; and we crossed with Ca 2+ -signaling knockout mutants with auxotrophic marker leu-3, and his-3 strains (Garnjobst, 1953;Adams et al., 1987;Coenen, 1994).

Strains and Growth Conditions
Growth and maintenance of Neurospora crassa strains on Vogel's medium supplement with glucose were essentially as described in Davis and De Serres (1970). The Ca 2+signaling knockout mutants, genotypes indicate as a Neurospora crassa unit number (NCU) (Table1, and Table  2). R.     The strains were obtained from the Fungal Genetic Stock Centre (FGSC), Kansas City, Missouri, USA. In this study we used two auxotrophic marker strains leu-3 and his-3 (leu-3, which is deficient to isopropylmalate synthatase, isopropylmalate dehydrogenase and isopropylmalate isomerase; his-3, histidinol dehydrogenase, phosphoribosyl-ATP-pyrophosphohydrolase and phosphoribosyl-AMP-cyclohydrolase) respectively; two auxotrophic strains leu-3 and his-3 were supplemented in the media with leucine (mg/ml) and histidine(mg/ml) nutrition for their proper growth. The crosses were performed by on Synthetic Crossing media 1X (SCM), and Sorbose-glucose-fructose media 1X (FGS) were used for germinating progeny/ascospore. The antibiotic hygromycin used at a working concentration 220 μg/ml for selecting Ca 2+ -signaling knockout mutants strains form their colonies.

Microscopic Analysis with Evan's Blue Staining
Sterile pieces of cellophane were spread on top of the surface of solid Vogel's glucose agar medium. Heterokaryons were enforced by co-inoculating conidia of two strains grown on the cellophane for 2 days. The cellophane contain hyphae was peeled off from the surface of the medium and stained with 1% Evan's Blue dye (GAFF, 1971;JACOBSON et al., 1998). The hyphae were stained for 15 to 25 minutes, and after that the cellophane was placed in a Buchner funnel containing a piece of filter paper pre-wetted with sterile water. Sterile water was then gently pipette over the cellophane and very weak vacuum was applied in order to clean off overload dye. The cellophane was then sited on a glass slide, with 5% glycerol to avoid drying, and observed under the bright field phase contrast microscope (Jacobson et al., 1998;Wu and Glass 2001;.

Results and Discussion
In primarily, we screened for heterokaryon incompatibility, heterokaryons homokaryosis and heterokaryons heterokaryosis of 20 Ca 2+ -signaling knockout mutants out of 48 merely in Yeast, Peptone and D-glucose (YPD) media, results showed het incompatibility. On this foundation, we crossed NCU05225, NCU06366, NCU06650, NCU07075, and NCU07966 Ca 2+signaling knockout mutants with the opposite mating types of leu-3 and his-3 auxotrophic marker strains (Table1 and  Table 2).
Here,'M' is 1kb (NEB) marker; 1(Wild types mating types 1m and 1M; mutant mating types 1a and 1A) 2, 3, 4, 5, 6 and 7 respectively. All probes obtained by gene specific PCR amplification with respective primers (except NCU06366, NCU06650 knockout probe, using primer hph R) 5F -5R, 5F -5R, 5F -hphR, 5F -hphR, 3F -3R, 5F -5R, and 5F -5R (Table4). Note that fragment resulting, respectively, from the digestion at the right and middle and the middle and left restriction sites of the intact gene is replaced in the disruption by the size of fragments duo to loss of the middle sites from the disrupted genes. Although the disruption bands is not seen as prominently in the DNA from wild type both mating types (1m and 1M), it is seen in its hygromycin-resistant mutant both mating types (1a and 1A).
Which revealed neither hyphal compartmentation nor cell death showed for the heterokaryon homokaryosis (Fig. 3, test: HO), whereas in heterokaryon heterokaryosis showed cell death. During the process of het incompatibility, some organelle modifications occurred like septal plugging, vacuolization, shrinkage of plasma membrane, organelle degradation, DNA fragmentation (Glass, 2006;Marek et al., 2003) (Fig.  1), and accumulation of lipid bodies are common microscopic features associated with het incompatibility. Blue staining retaining was indication of killing due to mating type associated het incompatibility ( Fig.3 and 4b, test: HO, test controls: C1 and C2, wild type control: C3), and live cells portion exclude the dye (Fig.3, test: HO). Both mating types are essential for sexual development, however, coexistence during the vegetative phase is lethal, and therefore display incompatibility ( Fig. 1) (SAUPE, 2000;Garnjobst, et al., 1956). The calcium signaling genes NCU05225 encode a 674aa residues of mitochondrial NADH dehydrogenase, NCU06366 encodes a 505aa residues of Ca2+/H+ antiporter, NCU06650 encode a of 186aa residues of novel Ca 2+ and /or CaM binding protein, a secretory phosholipase A2, NCU07075 encode a of 508 aa residues of CAX that is a Ca 2+ /H + exchange, and NCU07966 encodes a 1110aa residues of calcium transporting ATPase 3 (Table 1) respectively.
In additionally other two Ca 2+ -signaling genes encodes proteins like NCU02283 encode a 467aa residues of calcium/calmodulin-dependent protein kinase type I, and NCU09655 encodes plasma membrane zinc ion transporter(598aa) and phosphatidylinositol-4,5bisphosphate phosphodiesterase gamma 2(625aa)( Table 1) respectively. This calcium signaling genes encode protein might be involved in heterokaryons incompatibility process and further needs to be studied at the molecular level in N. crassa. Therefore, the result indicates suppression of mating type associated het incompatibility is not common phenomenon for all the Ca 2+ -signaling knockout mutants in N. crassa.

Conclusion
Previous reports suggest that the nuclei from two different genotypic strains are incompatible within the same cytoplasm (Garnjobst et al., 1956;Saupe, 2000). In N. crassa at least 11 het loci exist. Five of them, the mating type locus and het-c, -d, -e and -I were originally identified using forced heterokaryons between nearly isogenic strains. Here, we reported five Ca 2+ -signaling knock mutant strains ∆NCU05225, ∆NCU06366, ∆NCU06650, ∆NCU07075, and ∆NCU07966 play important role in the mating-typeassociated het incompatibility in N. crassa (Fig.2, test: HO, test controls: C1 and C2, wild type control: C3), additionally tested two more Ca 2+ -signaling knockout mutants NCU02283, NCU09655; in both the condition like heterokaryons homokaryosis and heterokaryosis of NCU02283, NCU09655 mutants display het incompatibility like the wild-type control (Fig. 4a, test: HO, test controls: C1 and C2, wild type control: C3). In N. crassa heterokaryon compatibility is shown by strains with identical genotypic class of progeny, whereas different genotypes of progeny are het incompatible. But the five Ca 2+ -signaling knockout mutant strains are het compatible suggesting that the knockout Ca 2+ -signaling gene may interact with the genes in het domain that result in induced mating type-associated het incompatibility in N. crassa( Fig.1.1 sup.info).
Therefore, the mutants of heterokaryons homokaryosis identical het loci specificity displayed development rate, normal conidiation, and aerial hyphae formation, suggesting that the mutation was compatibility (Fig.2, HO) and heterokaryotic homokaryosis auxotrophic mutant strains growth in limited nutrient condition. Whereas heterokaryons heterokaryosis different het loci specificity displayed decrease in the development rate like lack of growth, normal conidiation, and aerial hyphae formation, suggesting that the mutation was incompatibility (Fig. 2, and Fig. 4a, test: HO, test controls: C1 and C2, wild type control: C3).
During the time of experiment setup, we have taken more care in mixing of conidial cells from other conidial strains contamination, and we verified specific knockout mutants mating type as well. We reported here the involvement of Ca 2+ -signaling gene in heterokaryon incompatibility as a phenotypic expression in N. crassa not yet reported and further needs to be studied at the molecular level.
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