Senescence in fungi: the view from Neurospora

M I N I R E V I E W

Senescence in fungi: theview fromNeurosporaRamesh Maheshwari & Arunasalam Navaraj

Department of Biochemistry, Indian Institute of Science, Bangalore, India

Correspondence: Ramesh Maheshwari,

Department of Biochemistry, Indian Institute

of Science, Bangalore 560012, India. Tel.:

191 80 2334 1045; fax: 191 80 360 0814;

e-mail: [email protected]

Present addresses: Ramesh Maheshwari,

53/A2 4th Main, 17th Cross, Malleswaram,

Bangalore 560055, India. Arunasalam

Navaraj, Department of Hematology &

Oncology, University of Pennsylvania Medical

School, Philadelphia, PA 19104, USA.

Received 9 October 2007; accepted 31 October

2007.

First published online 18 December 2007.

DOI:10.1111/j.1574-6968.2007.01027.x

Editor: Richard Staples

Keywords

Neurospora ; aging; senescence; mitochondria;

mtDNA; plasmids.

Abstract

Some naturally occurring strains of fungi cease growing through successive

subculturing, i.e., they senesce. In Neurospora, senescing strains usually contain

intramitochondrial linear or circular plasmids. An entire plasmid or its part(s)

integrates into the mtDNA, causing insertional mutagenesis. The functionally

defective mitochondria replicate faster than the wild-type mitochondria and

spread through interconnected hyphal cells. Senescence could also be due to

spontaneous lethal nuclear gene mutations arising in the multinucleated myce-

lium. However, their phenotypic effects remain masked until the nuclei segregate

into a homokaryotic spore, and the spore germinates to form a mycelium that is

incapable of extended culturing. Ultimately the growth of a fungal colony ceases

due to dysfunctional oxidative phosphorylation. Results with senescing nuclear

mutants or growth-impaired cytoplasmic mutants suggest that mtDNA is inher-

ently unstable, requiring protection by as yet unidentified nuclear-gene-encoded

factors for normal functioning. Interestingly, these results are in accord with the

endosymbiotic theory of origin of eukaryotic cells.

Introduction

The ability to regenerate the hyphal tips continuously gives

the fungi the potential for indefinite growth, corroborated

by the discovery of a more than 1500-year-old fungal colony

of Armillaria bulbosa spread over a large forested area in

North America (Smith et al., 1992). Because of hyphal

fusions and perforated septa, the fungal colony is a cyto-

plasmic continuum in which aged or dysfunctional orga-

nelles – the nuclei and mitochondria – can be recycled and

replaced by the migration of functional copies from other

cellular compartments. Additionally, their multinuclear

condition enables any potentially deleterious mutant nucle-

ar gene to be complemented by its functional allele in other

nuclei. These unique features of fungi undoubtedly contri-

bute to their immortality. Therefore, the findings of senes-

cing strains in Neurospora spp. as well as a few other fungal

genera including wild strains of the ascomycete Podospora

anserina are of great interest. In these fungi, senescence is

promoted by the same features that otherwise contribute to

its immortality. For example, the dysfunctional mitochon-

dria containing the senescence-determining factor spread

throughout the mycelium and become dominant – a phe-

nomenon called suppressivity. Because genetic tools are best

developed in Neurospora, this fungus has considerable

promise as a model for investigation of mechanisms in aging

and death in higher eukaryotes.

The symbols used herein are as recommended by Perkins

et al. (2001). The wild-type gene is in three-letter lower case

italics with the superscript ‘1’; infrequently in two letters,

for example, the wild-type allele natural death is designated

nd1; and the mutant allele nd is in lowercase italics. Its

putative protein product is in nonitalic capital letters, e.g.,

ND. A heterokaryon is denoted by enclosing symbols of the

component nuclei, separated by a plus sign, in parenthesis;

as for example (sen1sen1). The mitochondrial mutants

are in brackets, for example (poky). The plasmid name

is in capital letters prefixed with a lower-case ‘p’, e.g.,

pKALILO, abbreviated as pKAL. The free autonomously

replicating pKAL is denoted as AR-kalDNA, and the

FEMS Microbiol Lett 280 (2008) 135–143 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

pKALILO inserted into mitochondria as mtIS-kalDNA

(Griffiths et al., 1990).

Discovery

In 1951, Sheng (1951) obtained a colony of Neurospora

crassa from the plating of UV-irradiated conidia that ceased

growth in successive transfers under all nutritional condi-

tions, whether grown on agar surfaces or in liquid cultures.

This mutant was named natural death (nd). It led to the

belief that senescing strains may be isolated from nature

for genetic investigations of the phenomenon of aging

and death that characterizes the development of all

eukaryotic organisms.

The first nuclear-gene senescing mutant from nature

was obtained by an unusual method: individual nuclei

from mycelium of wild Neurospora intermedia were

extracted in the form of uninucleate conidia (Navaraj et al.,

2000) and plated (see Fig. 3 in Maheshwari, 2005). Among

150 homokaryotic cultures, one showed senescence upon

subculturing. The mutant gene senescent was introgressed

into N. crassa through a series of nine backcrosses for

gene mapping and preservation in a heterokaryon (Navaraj

et al., 2000).

In 1965, E.L. Tatum’s group described mutants of

N. crassa symptomatic of senescence that arose during

routine transfers of cultures. These had fine hyphae lacking

visible flow of cytoplasm, showed ‘stop-start’ growth or

stopped growing altogether (Diacumakos et al., 1965; Garn-

jobst et al., 1965). Microinjection of mitochondrial prepara-

tion from the normal strains into the slow-growing strains

restored normal growth but microinjection of DNA from

nuclei had no effect, indicating that the abnormal pheno-

type is determined by mitochondria. The afore-mentioned

studies showed that: (1) senescence is due to nuclear – or

cytoplasmic (mitochondrial) mutations and (2) natural

populations are a source for obtaining potentially senescing

strains, either through sampling of conidia (Perkins &

Turner, 1988) or by plating soil containing ascospores

(Pandit & Maheshwari, 1996a) and (3) although initially

indistinguishable from an immortal strain, a senescing

strain is identified by cessation of growth in extended

propagation in long tubes (race tubes) or by serial transfers

in culture tubes using conidia (Griffiths & Bertrand, 1984;

Maheshwari et al., 1994). Senescence usually manifests in

five to 30 subcultures made at weekly intervals at room

temperature. A strain that has not senesced in some

50 subcultures is commonly regarded as immortal. In

Neurospora, senescing strains have been found in popula-

tions of N. crassa, N. intermedia and Neurospora tetrasperma

(Griffiths & Bertrand, 1984; Maheshwari et al., 1994;

Maas et al., 2005).

Distinction between senescence andother cell death phenomena

Senescence is the progressive loss of growth potential of

mycelium culminating in total cessation of growth when the

culture is considered as dead. The respiration of mycelial

suspensions or conidia of all senescing strains (‘Cytoplasmic

mutants symptomatic of senescence’, ‘Senescence caused by

mitochondrial plasmids’, ‘Senescence caused by nuclear

mutations’) measured as oxygen uptake switches from cya-

nide-sensitive cytochrome-mediated to a KCN-insensitive,

salicylhydroxamic acid (SHAM)-sensitive, alternate pathway

mediated by alternative oxidase and is associated with

deficiencies of cytochrome a and b. Because the hyphal cells

are interconnected through the septal pores, dysfunctional

mitochondria accumulate throughout the mycelium, result-

ing in displacement of normal mitochondria, and deficiency

of ATP (Pall, 1990). A single study on the ultrastructure of

plasmid-associated senescing hyphae showed vacuolization,

breakdown of nuclear and mitochondrial membranes, loss of

cristae and accumulation of dense material in the mitochon-

drial matrix (Bok et al., 2003). Senescence in fungi is distinct

from apoptosis as there is neither typical DNA fragmentation

nor the release of cytochrome c. Senescence is not equivalent

to autolysis because there is no hyphal fragmentation or lysis

of the cell wall. Senescence is perhaps not analogous to

necrosis, because there is no documented microscopic evi-

dence of loss of membrane integrity or release of cellular

constituents. Senescing mycelium of strains bearing pKALI-

LO showed large vacuoles (Fig. 1), as in the incompatible cell

fusions due to nonallelic het loci (Glass & Kaneko, 2003). An

expected alteration in senescing mycelium is extensive depo-

lymerization of actin-cytoskeleton.

The life span of strains collected from the same place may

vary (Table 1). However, the replicate subcultures from the

same parent stock die in about the same subculture. Interest-

ingly, the death of duplicate cultures after these had been stored

at � 15 1C for 12 months occurred in fewer subcultures,

indicating that degenerative changes continue even in the

frozen state (Maheshwari et al., 1994). One apparently normal

strain did not resume growth after storage. Subsequently, it was

found that the strains tested contained a circular mitochondrial

plasmid (D’Souza et al., 2005a). The results of this experiment

suggest that senescence in fungi is an expression of a prepro-

grammed pathway that is kept in check in the long-living

(‘immortal’) strains by mechanisms that are not understood.

Distinguishing between nuclear andcytoplasmic senescence

Whether the senescence-determining factor resides in the

nucleus or in the mitochondria can be determined from

genetic methodology.

FEMS Microbiol Lett 280 (2008) 135–143c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

136 R. Maheshwari & A. Navaraj

Genetic cross

When two Neurospora strains of complementary mating

types, mat A and mat a, are crossed, the progeny inherit the

mitochondria only from the protoperithecia-forming

(female) parent. In reciprocal crosses: (1) long-living female

X short-living male, progeny is long living whereas in

(2) short-living female X long-living male, progeny is

short living.

Heterokaryon test

A mycelium containing nuclei from a senescing and a

nonsenescing strain is constructed by mixing germinating

conidia of a long-living strain with that of a senescing strain.

The neighboring cells of two genetically related strains

interconnect, and the heterokaryon containing mixed cyto-

plasm grows indefinitely on minimal medium due to the

complementation of nuclei from the original strains. Color

and auxotrophic markers are incorporated into the fusing

strains by prior genetic crossing for detection of the hetero-

karyon (Fig. 2). The short-living or long-living nuclear types

are extracted by plating conidia formed by heterokaryotic

mycelium on appropriate media.

Cytoplasmic mutants symptomatic ofsenescence

A class of cytoplasmic mutants exhibit alternate rapid

growth, followed by slow or no growth on agar media

(Bertrand et al., 1980; de Vries et al., 1986; Almasan &

Mishra, 1988, 1990). The mtDNA of these mutants, for

example (stopper), have deletions and exhibit severe defi-

ciencies of cytochromes b and aa3 (Almasan & Mishra,

1991). The defective mitochondria are maintained in a

heteroplasmic state with normal mtDNA, as in several

human diseases (Wallace, 1999). In Fig. 2, has been illu-

strated a technique for preserving lethal nuclear mutants in

heterokaryons. As long as nd or sen is associated with a

normal allele in other nuclei of the heterokaryon, it is

indefinitely stable. However, its mtDNA undergoes rapid

deletions when removed. Cloning and sequence analysis of

deleted fragments in nd and comparison with wild-type

mtDNA revealed high frequencies of deletions of interven-

ing sequences of the mitochondrial genome due to unequal

crossovers between mispaired copies of mtDNA sequences.

The nucleotide sequence of sen-specific EcoRI-5

fragments suggested that intramolecular recombination

between stretches of 18 nucleotide palindrome sequences

(50-CCCTGCAGTACTGCAGGG-30) in mtDNA that poten-

tially form hairpin structures could promote preferential

intramolecular recombination (Dieckmann & Gandy, 1987;

Almasan & Mishra, 1991) between homologous repeats,

resulting in deletions (Fig. 3). The studies of growth mutants

of fungi including the yeasts (Contamine-Picard & Picard,

2000) have revealed the innate instability of mtDNA, and the

requirement of nuclear gene-encoded proteins for main-

taining the integrity of mtDNA.

Table 1. The number of passages (transfers) of agar-grown stock

cultures of four strains of Neurospora intermedia before death�

Strain

Number of passages before death

% Reduction

in life-span

Before

storage

After 12 month

storage at � 15 1C

(Series I) (Series II)

Maddur 1991–3 25 17 32

Maddur 1991–59 29 14 52

Maddur 1991–60 15 12 20

Maddur 1992–18 29 9 69

�Subculturing by asexual spores (conidia) or vegetative hyphae on slants

was at intervals of 5–10 days. From Maheshwari et al. (1994).

Fig. 1. Mycelial morphology of Neurospora

crassa. (a) A long-living wild-type strain.

(b) Senescing subculture of pKALILO bearing

strain after three subcultures. Scale bar 100 mm.

From Bok et al. (2003). Reprinted with

permission The Mycological Society of America,

from Mycologia.


137Senescence in Neurospora

Senescence caused by mitochondrialplasmids

The determination whether growth-impaired mutants

will arise upon prolonged cultivation from the ‘normal’

strains, as the spontaneously occurring petite mutants in

yeast, led to isolation of maternally inherited (poky)

(Mannella et al., 1997) and stopper (stp-B1) mutants.

Neurospora intermedia cultures isolated from nature were

subjected to extended culturing either by continuous

vegetative growth in race tubes or sequential conidial

transfers in slants (Griffiths & Bertrand, 1984). Approxi-

mately 30% of the strains from Kauai were senescent in

prolonged culturing and were named ‘kalilo’, Hawaiian for

‘dying’. Senescing strains of N. crassa were found in the

state of Maharashtra in India, and were named ‘maranhar’

meaning ‘prone-to-death’ in the Hindi language (Court

et al., 1991). Both kalilo and maranhar strains contain

linear mitochondrial plasmids that have no nucleotide

sequence homology.

The circular plasmids pVARKUD (3675 bp) and pMAUR-

ICEVILLE (3581 bp) contain an ORF encoding a reverse

transcriptase. From the sequence data of mtDNA and

plasmid DNA, it was deduced that the retroplasmid inserts

into the mtDNA. The insertion is affected by prior forma-

tion of a variant plasmid form having a cDNA copy of a

tRNA-like cloverleaf structure at the 30 end (Akins et al.,

1989). The variant plasmid DNA integrates by homologous

recombination into mtDNA, close to or within the genes

encoding the mitochondrial rRNAs (Myers et al., 1989;

Chiang et al., 1994). For unknown reasons, the mitochon-

dria containing the variant plasmids divide faster than the

wild type; the presence of nonfunctional mtDNA molecules

exceeding a threshold concentration in obligate aerobes is

expected to cause drastically lowered ATP production and to

result in death (Myers et al., 1989). Although Akins et al.

(1986) found that impaired growth and cytochrome defi-

ciencies correlated with the time of insertion of variant

plasmid into mtDNA, Fox & Kennell (2001) found no

correlation between the time of insertion of plasmid se-

quence and its suppressive accumulation (Stevenson et al.,

2000). Insertion of pKAL (8642 bp) and pMAR (7052 bp)

into mtDNA was correlated with disruption of the nuclear

membrane and vacuolization in the mitochondrial matrix

(Bok & Griffiths, 2000).

Spread of plasmids

Twenty-five years after the initial discovery, a resampling of

strains from Hawaii by Southern hybridization using plas-

mid fragments as probes showed that nonsenescent and

senescent fungal strains coexist. pKAL was present at the

Fig. 2. Preservation and use of a recessive lethal

nuclear gene mutant of Neurospora crassa.

Diagram of construction and resolution of

(sen1sen1) heterokaryon (the mycelium is

represented by rectangles; nuclei by open

circles; and mitochondria by ovals. Closed circles

or ovals represent sen1; open circles or ovals

represent sen). Heterokaryons grew on minimal

medium and produced orange conidia. The

heterokaryons were resolved by plating

macroconidia. White-conidiating cultures that

grew on nicotinamide-supplemented medium,

but not on minimal or pantothenate medium

were tested for senescence. The heterokaryons

and the resolved sen1 cultures were analyzed for

mtDNA deletions.

Fig. 3. Diagram of recombination events in sen mtDNA illustrating the

formation of sen-specific EcoRI fragments of sizes 3.9, 4.4 and 3.6 kb as

a result of recombination events between fragments 10 and 5 (junctions

J1 and J3 or junctions J2 and J3) or between EcoRI fragments 10 and 8

(junctions J2 and J4). Gene symbols: ndh, NADH dehydrogenase; cyt,

cytochrome; urf, unassigned reading frame; CO, cytochrome oxidase.

From D’Souza et al. (2005b).



same frequency, i.e., c. 30% (Maas et al., 2005). pKAL has

also been found in N. tetrasperma, although at lower

frequencies than in N. intermedia. Senescing isolates have

also been identified in populations of Neurospora collected

in Maddur in southern India (Maheshwari et al., 1994;

D’Souza et al., 2005a). Time-course analysis revealed inte-

gration of pMAD into mtDNA. All cultures collected from

Maddur were senescent and contained pMAD that was 98%

homologous to pMAU first found in the Mauriceville strain

of N. crassa collected from Texas. This raises the question as

to how plasmids have become distributed in geographically

separated regions. Because mitochondrial leakage during

paternal transmission is thought to be rare, horizontal

transmission of senescence plasmids through hyphal fusion

has been suggested for the widespread distribution of

senescence plasmids, despite the presence of polymorphic

het loci that minimize, if not preclude, hyphal fusion. To

test transmission of the senescence plasmid into a nonse-

nescent colony, a senescing kalilo strain having the nuclear

marker genes nic-1 al-2 was fused to a long-living strain

having the nuclear marker genes ad-3B cyh-1 (Griffiths et al.,

1990). The resulting heterokaryon was senescent in which

mtIS-kalDNA had associated with the nuclei. Evidence

has been provided indicating that hypha can fuse and

form heterokaryotic mycelium when growing inside a

natural substrate (Pandit & Maheshwari, 1996b). The spread

of plasmids by vegetative fusion of hyphae is therefore a

real possibility.

To determine whether plasmids confer some advantages

to the host strain, a kalilo plasmid from a nonsenescent

N. tetrasperma was introgressed into wild-type N. crassa.

The growth rate at 41 1C (Tmax) and fertility (perithecia

production) at 30 1C (4Topt) improved (Bok & Griffiths,

2000), suggesting that plasmids influence house-keeping

genes in the host fungus, hastening its life cycle. Presumably,

a plasmid-bearing strain initially starts out as more fertile

than the strain lacking plasmids. According to the selfish

gene theory of Dawkins (1989), the host fungus is a vehicle

for propagating parasitic DNA; the plasmid is not only

selfish DNA but true parasitic DNA, spreading by contact

transmission like a molecular disease.

Temporary increase in life span

Not every culture that carries pKALILO senesces; for exam-

ple 10–20% pKAL carrying isolates of N. intermedia, and a

similar percentage in N. tetrasperma isolates, did not senesce

(Maas et al., 2005). Crossing of these strains to nonsenescing

strains showed that the ability to tolerate a potentially lethal

plasmid is maternally inherited. When short-lived strains

were intercrossed prior to the terminal stage, the sexual

progeny became temporarily rejuvenated (Griffiths & Ber-

trand, 1984; Maheshwari et al., 1994). A plasmid trapping

approach demonstrated that a cryptic mitochondrial retro-

plasmid in the strain interacts with a senescence-inducing

plasmid (e.g. pKAL) to form hybrid plasmid, suppressing

the detrimental effect of the senescence-causing plasmid

(Yang & Griffiths, 1993; Maas et al., 2007). Although

horizontal transmission of senescence-causing plasmid

can occur, the fact that Neurospora populations are

sexual (Pandit & Maheshwari, 1996a) suggests that mecha-

nisms operating during the sexual phase suppress the

plasmid spread.

Senescence caused by nuclear mutations

A recessive lethal nuclear gene mutation can be maintained

in multinuclear fungi – masked in its expression by its wild-

type allele in other nuclei – until segregated at the time of

asexual spore formation. The homokaryotic mutant spore

may produce an exceptional unstable mycelium. In this

context, the isolation of a single nuclear-gene, mutant

senescent, isolated from single uninucleate microconidia

formed by a phenotypically normal wild-collected strain

of Neurospora using microconidia assumes importance

(Maheshwari, 2005). Mutant senescent is similar to mutant

nd described earlier. Death occurs in six to nine subcultures

at 26 1C, but in only two subcultures at 34 1C (Navaraj et al.,

2000). To avoid permanent loss of genotype, nd and sen are

maintained as heterokaryons from which they are extracted

by conidial plating when desired.

Measurements of oxygen uptake of conidia or mycelial

homogenates or germinating conidia of nd (Seidel-Rogol

et al., 1989) or sen (Navaraj et al., 2000) using respiratory

inhibitors, and the analyses of mitochondrial cytochromes

by measuring difference spectra, revealed that in cultures

grown at 34 1C, cytochromes b and aa3 were present but

cytochrome c was absent. By contrast, at 26 1C, cytochromes

b and c were present but cytochrome aa3 was diminished in

the late subcultures. However, the deficiency of the respira-

tory chain cytochromes may not be the primary cause of

death of the sen mutant because the cytochrome c and aa3

mutants of N. crassa are capable of sustained growth

whereas sen is not. A comparison of EcoRI restriction digests

of dying nd (Seidel-Rogol et al., 1989) and sen with long-

living wild-type N. crassa mtDNA showed deletions and

gross sequence rearrangements (D’Souza et al., 2005b),

resulting from suppressive forms of defective mtDNA mole-

cules that are generated by defects in mtDNA maintenance

due to mutations in the nuclear gene. The two gene products

ND and SEN have mutually exclusive roles in the main-

tenance of mtDNA in N. crassa, presumably in protecting

certain hyperactive recombination regions from cleavage by

endonucleases. It is not proven, however, whether PstI

palindrome is involved in the generation of deletions

(Bertrand et al., 1993).



Thermosensitivity

The nuclear gene mutants, nd and sen, die faster at 34 1C

compared with 26 1C (Fig. 4). Vigor is not regained if

cultures initiated at 34 1C are shifted to 26 1C. Because

respiration is critical for viability of Neurospora, nuclear

genes – essential for cell survival – could only be identified

when recovered as heat-sensitive mutants. The heat-sensi-

tive mutants named unknown for unknown requirements

(see Perkins et al., 2001) have restricted growth at 34–37 1C

but are normal at 25 1C. These mutants may be deficient in

components of multiprotein cytochrome aa3 (Nargang

et al., 1995), or of mitochondrial outer membrane and inner

membrane translocase. For example, in N. crassa, tom22,

tom40 and tom70 encode protein components of translocase

of the outer mitochondrial membrane (TOM) and the

growth of these mutants is severely affected at elevated

temperatures (Harkness et al., 1994; Nargang et al., 1995;

Grad et al., 1999). The indispensability of a nuclear-encoded

component of the mitochondrial TOM could be recognized

through use of a genetic trick called the sheltered-RIP, which

allows a mutated indispensable allele to be maintained in a

heterokaryon with a functional copy of the gene in another

nucleus – the culture thus remaining viable. Modification in

nucleus-encoded proteins of the mitochondrial import

apparatus would be expected to have pleiotropic effects as

the translocase complexes are involved in assembly of

mitochondrial and enzyme proteins of the electron trans-

port chain, mtDNA stability assembly of ribosomes, and

transport of a number of small molecules (adenine nucleo-

tides, inorganic ions, amino acids, fatty acids) across the

inner membrane. In yeast, the disruption of mitochondrial

carrier protein components resulted in severe growth defects

(Contamine-Picard & Picard, 2000). These observations

suggest that modifications of the mitochondrial import

apparatus impair the amounts of nucleus-encoded proteins,

affecting the stability of the mitochondrial genome and life

span. In P. anserina, mutations in genes that encode proteins

of the mitochondrial TOM complex resulted in accumula-

tion of defective mitochondrial genomes (Jamet-Vierny

et al., 1997).

How might the temperature sensitivity of sen be ex-

plained? We speculate that SEN protein synthesized in the

cytosol is an essential component of the multiprotein TOM

complex, involved in recognizing proteins made on cytoso-

lic ribosomes, and their import into mitochondria (Neu-

pert, 1997; Kunkele et al., 1998). Depending on the growth

temperature, SEN assumes two metastable structures and is

inserted into the mitochondrial membrane (Fig. 5). Once

the mutant form of SEN is trapped in the mitochondrial

membrane, conformational interconversion is unaffected by

temperature. It is likely that several of the thermosensitive

nuclear genes, presently classified as unknown (Perkins et al.,

2001), encode protein components of the mitochondrial

translocase mediating the recognition and transfer of cyto-

solic-made preprotein into mitochondria. An implication of

loss of cytochrome is the intracellular signaling known as a

retrograde response (Jazwinski, 2000) and expression of the

cyanide-resistant alternative oxidase pathway, which

branches from the cytochrome pathway at ubiquinone, an

expression that is insufficient to reverse the senescence. The

oxidative stress could lead to production of reactive oxygen

species that are postulated to attack and damage proteins,

lipids or DNA – providing a genetic and biochemical link in

senescence.

Outlook

Researches on senescing strains will help to understand the

origin of plasmids, their maintenance and spread in vegeta-

tive cultures and their elimination during the sexual phase.

Research should further the identification of structural

regions in the mitochondrial genome and the enzymes

involved in recombination that can lead to deletions

that form plasmid-like elements (Almasan & Mishra, 1990;

Hausner et al., 2006).

The nuclear-gene senescing mutants are particularly

valuable in the appraisal of the endosymbiotic theory.

According to this theory, a eukaryotic cell evolved from

the engulfment by a wall-less archaebacterium, following

which an endosymbiotic relationship evolved, accom-

panied by the transfer of most of the bacterial genes

Fig. 4. Growth rate of senescence. Subsets of ascospore progeny

cultures from a cross wild-type X heterokaryon (sen1am1 ad-3B) were

grown to conidiation at 26 and 34 1C. From the two sets of cultures at 26

and 34 1C, the original and the subsequent sen cultures were inoculated

on an agar medium in race tubes and grown at 22 1C (previously at

26 1C, 1–6) and 34 1C (I and II). From Navaraj et al. (2000).



into the host nucleus. A consequence of this evolutionary

relationship is that in the present-day eukaryotic cell,

the preproteins translated on the cytosolic ribosome are

imported into the mitochondria via protein complexes

located in the outer and inner mitochondria mem-

branes. The natural populations of Neurospora are a valuable

resource for obtaining both nuclear and extranuclear

mutants. These mutants will be useful for identifying

additional loci involved in the maintenance or replication

of the mitochondrial genome and the interdependence

between the nuclear and mitochondrial genetic systems.

It is expected that with the availability of the genome

sequence of N. crassa efforts will be made to identify ND

and SEN.

Acknowledgements

Research in R.M. laboratory was supported by the Depart-

ment of Science & Technology, Government of India.

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Senescence in fungi: the view from Neurospora

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