INTRODUCTION TO LATE BLIGHT AND IMPORTANT POTIENS AND GENES

EXPRESSED DURING EARLY HOURS OF INTERACTION OF

PHYTOPHTHORA INFESTENS ASSOCIATED WITH LATE BLIGHT OF
POTATO
Malka Arif

Institute of Agriculture Sciences, Punjab University.

Abstract:

Following article presents a general introduction to Late Blight and an overview of genes
and gene products which are proved to play important role in host-pathogen interaction of
Phytophthora infestans with potato. It include pihmp1, a gene encoding haustorium specific
membrane protein, six genes extracted from cDNA library of induced mycelium (IM) of
P.infestans, which were upregulated at the initiation and during the infection; and gene
products (proteins) extracted from apoplast during compatible and incompatible
interactions of P.infestans with potato. Possible candidates for the initiation of
hypersensitive response and extracellular effectors (source of ETS, effector triggered
susceptibility) were discussed.

INTRODUCTION:

In terms of human consumption, after Rice and wheat, Potato (Solanum tuberosum L.) can
be designated as third most popular food crop in the world. Above a billion people
consume potatoes worldwide, and in 2014 the global potato production surpassed 382
million metric tons (FAO, 2017). In annual report of 2015, Pakistan Bureau of Statistics
reported the production of 27,43,300 tons and an area of 148600 hectares under potato
cultivation in year 2013-14 (Bureau of Statistics, Government of Pakistan)

Phytophthora infestans, the causal agent of Late Blight of Potato is a globally well
recognized devastating pathogen responsible for annually 16% global yield losses while
the global cost of damage and control is estimated about €109 by Haverkort et al. in 2008,
2009, while Haas et al. in 2009 reported annual losses of 6.7 billion on global scale due to
this pathogen. In history of plant pathology, P.infestens is quite infamous as the cause of the
“Irish potato famine” in 1845 that became a reason of above a million fatalities and
migration of even more (Beyer K. et al, 2002). As preventive measure a heavy spray of
fungicides is currently in use. However, an enormous usage of these chemicals is now
posing serious threats to environment and human health. Additionally, some commonly
used chemical agents can cause insensitivity in fungal isolates (Goodwin et al., 1996).

Another important control measure is to plant potato cultivars resistant to P.infestans.

There are two main sources of inducing resistance in potato varieties against this pathogen,
one is ‘Qualitative resistance’ and other is ‘Quantitative resistance’. Qualitative resistance
which is conferred by R-gene (or genes), in accordance with the “gene-for-gene” model by
Flor in 1971. As a result of this interaction Effector-triggered immunity is stimulated that
leads to the activation of HR (hypersensitive response) as stated by Kamoun et al. in 1999;
and by Jones & Dangl in 2006. Such type of interaction is termed as Incompatible
interaction while such a fungal isolate is called avirulent (Lebecka R. & Sobkowiak S. 2013).
For single race-specific resistance (R) genes, the wild solanum specie, Solanum demissum
has provided resistant genes, which can confer thorough resistance to avirulence protein
carrying P. infestans isolates (Lenman et al., 2016). However, the pathogen can evolve
rapidly because of a dynamic, repeat-rich domain of genome (Raffaele et al., 2010; Haas
et al., 2009) and hence has led to overthrow resistance of host through development of new
pathogen races capable of infecting previously resistant Potato cultivars (Fry & Goodwin,
1997). Pyramiding of multiple R genes is now advised to be a suitable strategy for a longer
period maintenance of plant’s resistance (Haesaert et al., 2015; Zhu et al., 2012)

Quantitative Diseases Resistance(QDR) or general resistance, as a result, is now being used

in breeding programs to replace short duration R- gene mediated Resistance (St. Clair,
2010). QDR can also be explained as horizontal, broad-spectrum, durable, field resistance
and incomplete resistance controlled by multiple genes (Poland et al., 2009; Solomon
Blackburn et al., 2007). QDR often leads to reduced severity and symptoms but not
complete absence of disease. Such resistance is considered more durable and the reason lie
in reduced evolutionary pressure on pathogen for adaption. Therefore, QDR is preferred by
breeders of potato ever since 1960s piling of R genes was easily overthrown as shown in
the cultivar Pentland Dell for R1, R2, and R3a (Hein et al., 2009).

The gene study presented in this article may help plant researchers in selection of a
suitable gene or gene product, during early hours of infection, for the development of a
plant resistant to P.infestans, such that minimum or no symptoms appear. However, this
article only provides a brief overview of genes confirmed to play an important role in early
infection hours and a detailed study will be required to choose a crucial gene or
combination of gene to provide efficient quantitative resistance.

HISTORY OF LATE BLIGHT:

P.infestans was probably the first fungi in history of plant pathology that acquired
worldwide attention as it wreaked havoc in potato fields of United States & Europe and
caused some serious epidemics. Late Blight of potato first appeared in America at a
noticeable level in 1843 while in Europe, England and Belgium it appeared in 1844 and had
reached in Netherlands next year by the June. The rate of its spread was alarming. Ireland
was affected most severely, mainly because of their sole dependence on potato crop back
then. The rural Irish community was poor and had almost nothing to eat but potatoes
(Widmark A.K, 2010). After the ‘Great Irish Famine’ of 1846-1848 an extensive amount of
research was focused on cause of this ‘fatal malady’ of potato (as referred by people at that
time) that has destroyed the fields. This period signifies the origins of plant pathology by
the work of Antone De Barry and Reverend Berkeley. In 1876, Barry provided a strong
evidence in the favor of “Fugal theory” of Late Blight and argued that the blight is caused by
a fungus. He named the fungus as Phytophthora infestans. These scientists also advocated
that the fungus was not indigenous but rather brought into Europe from Andes of South
America. In 1939, however, Reddick presented another concept, which now is more widely
accepted; that Mexico might be the center from where late blight was originated (Z. Gloria
Abad & Jorge A. Abad, 1997). The reason of this lies in the facts that; Only Mexico contains
both mating types of P.infestans in 1:1, gene for gene relation exists between P.infestans
strains and some wild Mexican varieties of Potato and the area has a wide variety of
phenotypic and genotypic characters of Late Blight. In spite of above facts, South America is
still considered to be a potential candidate, based on recent researches (Widmark A.K,
2010).

TAXONOMY OF PHYTOPHTHORA INFESTANS:

Phytophthora is a genus of kingdom Stramenophila and phylum oomycota. These are a

category of microscopic organisms which appear to be fungi physically, but are more
relevant to aquatic organisms such as diatoms, brown and golden-brown algae etc (Dick,
2001; Förster, 1990). Taxonomy of P.infestans is a very controversial subject. This
organism has successfully eluded taxonomist ever since its discovery and even now a
number of taxonomic trees exists.

BIOLOGY OF PATHOGEN:

The hyphae of Pthytophthora are non-septate (coenocytic) in comparison to true fungi

which have septated mycelium. The genus Phytophthora contains more than 60 species
which are majorly parasitic (Erwin & Ribeiro, 1996). During a larger part of their life-cycle
the oomycetes are diploid (2n). They contain Cellulose and other glucans in their cell wall
instead of chitin as in true fugi (Bartnicki-Garcia, 1968). Members of this Genus are not able
to produce thiamine and sterol (Ribeiro & Erwin, 1996), and thus have to obtain these
important materials from their host. This fungus is a pathogenic Hemibiotroph and
possesses a relatively narrow host range. Most of its hosts belong to the plant genera
Solanum and Lycopersicum (Ribeiro & Erwin, 1996). The two main crop hosts are potato
and tomato (Solanum lycopersicum L. or Lycopersicum esculentum)

SYMPTOMS OF LATE BLIGHT:

Initial symptoms of potato late blight in the field consists of small, circular to irregular
watersoaked spots which are dark to light green in color. During moist and cool weather
these small spots coalesce and fuse to form large black or brown greasy lesions which are
turned into patches. In wet weather white mildew like area also appear on edges of lesions
while in dry warm weather the patch become dry. Soon the whole leaf is covered with such
patched of dead tissue and is killed in few days. Extensively infested fields give off a distinct
odor of rotted tissue.

Tubers infection is evident by formation of irregular areas of purple to brown color.

Reddish brown rot is present in underside of potato skin (Kumar et.al., 2017)

LIFE CYCLE OF PATHOGEN:

Asexual stage:

As described by F. Govers et al. 1997, when potato tubers are planted in field during
spring, sprouts arise from tubers which are infected with P.infestans mycelium. In case of
favorable conditions sporangiophores appear from stomata releasing many airborne
spores and a rapid advance of disease is anticipated. Usually above 12-15∘ C sporangia may
germinate directly while below 12∘ C sporangia give raise to many motile zoospores which
germinates after encystment. Both zoospores and sporangia have ability to infect the plant
but zoospores are considered more important.

On leaf & stem surfaces sporangia and cysts germinate and form germ tube with
appressoria, from which penetration hyphae (haustoria) are formed. From epidermal cells
the mycelium grows intercellularly at first and then intracellular haustoria penetrate
mesophyll cell layer. Under favorable conditions, sporangiophores carrying sporangia are
formed with in 4 to 5 days after infection. Sporangia are either dispersed by air or washed
down into the soil by rainwater. Spores germinate in soil and infect the tubers through
lenticels, wounds or eyes. Mostly such tuber rot either in field or in storage but few
survives the winter to produce primary inoculum in next season.

Sexual stage:

Fungus P.infestans is heterothallic and thus has two mating types, A1 and A2. When the
hyphae of A1 and A2 come in contact, antheridia (male sex organ) and oogonia (female sex
organ) are generated. An oospore is formed as a result of fertilization, and as it matures a
thick wall develops around oospore. Oospores of Oomycota are very resistant structures
and thus can survive in soil for several years.

TRANSMISSION OF PATHOGEN:

Late Blight is primarily is a tuber born disease. The mycelium, spores and zoospores are
short lived and require living plat material to survive non growing period in field. Most
commonly this plant material is potato tubers left in field and cull piles. As a result both
quality and quantity of tubers is reduced. In contrast to above asexually produced
structures, if both mating types exist in a field, sexually produced oospores are formed.
These oospores are very resistant structures and require no host to survive in soil.
STATUS OF LATE BLIGHT IN PAKISTAN:

The first ever occurrence of late blight was reported in 1984 in Sawat valley (Majeed et.al.,
2014) Ever since then a considerable amount of efforts have been put on occurrence and
management of Late Blight of potato and Tomato. In Pakistan P.infestans affects potato
fields in Punjab, the major potato growing area, NWFP and also in some areas of
Baluchistan. The yield losses due to Late Blight are severe, ranging from 50 to 100% in
some areas when season become highly favorable. In January 2018, Abdul kareem
published a report in which he claimed occurrence and serious attacks of Late Blight of
potato in fields of Gilgit Baltistan. It was reported that P.infestans have been isolated from
all the potato growing areas of Gilgit Baltistan and have also been found to affect all growth
stages of tubers, stems and leaves of potato plant. Presence of A2 mating type (Ahmed I. &
Mirza J.I, 1995) and Matalaxyl (widely used fungicide against P.infestans) resistant isolates
of P.infestans has aggravated the situation (Ahmed I. et al., 2012). Currently all commercial
cultivars grown in Pakistan are susceptible to Late Blight of Potato (Iftikhar Ahmed et. al.,
A. Kareem, 2018).

IMPORTANT POTIENS AND GENES EXPRESSED DURING EARLY HOURS OF

INTERACTION OF PHYTOPHTHORA INFESTENS:

Gene Coding Haustorium Specific Membrane Protein:

Avrova A.O. et al in 2008 reported P.infestans gene cloning which they named Pihmp1, from
a suppression subtractive hybridization SSH library, that codes for a glycosylated protein.
This protein has four closely packed transmembrane helices. An upregulation in the
expression of Pihmp 1 was observed in germinating cysts and also in germinating cysts
with appressoria and was considerably upregulated during whole process of infection of
potato. Translation of Pihmp 1was evident in infection structures such as in germinating
sporangia, germinating cysts and appressoria. Importantly was observed to accumulate in
the appressorium, and during infection was found at the haustorial membrane. During the
infection of potato, Pihmp1 was observed to localize in the haustorial membrane of
P.infestans. It was also discovered that it takes about a period of 3 h for haustorial
structures of P. infestans to form over, mature in about 12 h. They proposed that on the
surface of intercellular hyphae formation of these structures is stimulated only at locations
where Pihmp1 is localized. It was proposed that “Pihmp1 is an integral membrane protein
that in infection structures, provide physical stability to the plasma membrane of P.
infestans”. Also a loss of Pathogenicity was observed when gene Pihmp1 was silenced. The
loss of pathogenicity shows possible participation of gene in processes of penetration and
early infection by P.infestans.
Genes isolated from Induced mycelium:
Beyer K. et al. in 2002 provided first evidence of presence of proteins specific to biotrophic
structures such as infection vesicles or haustoria, on the surface of oomycete haustoria,
which are necessary for successful host colonization by P.infestans.

The team made a successful attempt of isolating the genes which were expressed during
some of the initial steps of interaction. To induce the mycelium of P.infestans, it was
brought in touch with potato plant and after that mycelium was detached from tissues of
plant. A comparison of this induced mycelium with a mycelium of P.infestans incubated in
water was carried out through generating a differential cDNA library by SSH. In the dot blot
analysis, while transcript levels of numerous cDNA fragments were enhanced; only the six
of the cDNAs clones were picked for further study.

1. IM-002A: Amino acid transporter

2. IM-003C: Sucrose transporter
3. IM-OO4A: No clear homology
4. IM-008B: Spliceosome associated protein
5. IM-01F1: Abc-transporter
6. IM-06D11: CDC 6

IM-002A, IM-003C, and IM-008B express almost same pattern of expression. The
expression of these genes was observed majorly in mycelium, sporangia and in the infected
tissue of plant. The expression of IM-002A, however, was shown only in cysts & zoospores.
On the other hand, genes IM-008B & IM-003C were not expressed in these structures.
IM008B also exhibit a comparatively high expression in germinated cysts. The expression
pattern of IM-004A (clear homology of which has not been found in the database) matched
to that of Actin gene (Actin gene or ‘actA’ of P.infestans, is a gene which is expressed in all
developmental stages tested at almost same levels as stated by Unkles et al. in 1991). IM-
01F1 was expressed in all stages, but its expression levels in mycelium of pathogen and in
plant material infected with mycelium, showed obvious upregulation. IM-06D11 was
mainly expressed in zoospores and cysts, which are important infectious agents, and also in
mycelium.

After 8hr incubation in water, the first detectable expression of almost all the selected
genes appeared 3 days after infection, and after that transcript levels increased
continuously. Here two important genes, IM-06D11 & IM-01F1 showed elevated expression
levels in planta; than in vitro. Highest expression level for IM-01F1 was at 5 days after
infection, which was about two fold greater than its expression in in-vitro mycelium. IM-
06D11 was suggested probably the only gene clone that clearly showed expression at the
first detectable time point after infection which was at Day 3 AI. At Three days post
infection expression level of this gene was four fold higher as compared to its expression in
vitro mycelium, afterwards the transcript level of IM-06D11 decreased slowly. Therefore it
was suggested that this gene could be significant during early time points of infection
process.

Apoplastic Protein/ Transcript Isolates:

To have a comprehensive molecular understanding of late blight of potato, in 2014, Ali A.
et. al studied interaction of Phytophthora infestans with compatible and incompatible
potato cultivars. They selected the two resistant potato cultivars SW93-1015 and Sarpo
Mira and a non-resistant (or compatible) variety Desiree along with pathogen P.infestans
SE-03058. They analyzed quantitative proteomics of 51 apoplastic secretome samples at
different time points such as at 6, 24 and at 72 hours after inoculating with pathogen strain.
The object was to detect changes taking place abundance in protein and expression of
genes during interactions.

Protein components expressed during non-compatible interactions only, were considered

responsible of HR induction. In potato, genes associated with the induction of HR are
poorly understood. Among few of many genes expressed in both resistant potato cultivars
include MYB transcription factors, glutaredoxins, and a zinc fin-ger protein encoding genes.
It is maybe important to note that plants can become resistant to the necrotroph Botrytis if
they have defective glutaredoxin activity. (La Camera S. et al. 2010). A protease inhibitor
similar to Kunitz, transcription factors and a protein similar to RCR3 were also seemed to
be associated with HR. During incompatible interactions however, Plant U-box proteins are
involved which consists of a family of genes which have multiple roles and is associated to
PAD4 and salicylic acid mediated pathways (Vogelmann K. et al. 2012). Based on this
extensive apoplast proteomics data, Ali A. et al also published a table of possible Candidates
for hypersensitivity initiation.

Effector-triggered susceptibility (ETS) is the suppression of plant defenses through

effector molecules of pathogen that leads eventually to its compatible interaction with
host. Extracellular effectors of P.infestans are inhibitor molecules which target host’s
proteins which are related to defense; for example proteases and glucanases, and other
defense related mechanisms such as HR. Just as EPI1 and EPI10; two extracellular serine
protease inhibitors similar to that of Kazal, act to inhibit a serine protease like subtilisin
P69B, in tomato plant (Tian M. et al. 2004). These two genes also bind & inhibit the
cysteine protease C14. Effector-target C14 protease is a well characterized gene. It is a
papain-like cysteine protease which is an easy target for two apoplastic, cystatin-like
proteins secreted by P.infestans, EPIC1 and EPIC2B. More importantly, silencing
of C14 increase susceptibility of potato to P. infestans, indicating that this protease plays a
crucial role in pathogen defense (Kaschani F. et al 2010).

Due to the fact that during compatible interaction some specific proteins in the cell’s
apoplast were suppressed selectively, but not the incompatible interactions, lead to the
screening of putative effector targets. Important candidates were single proteins from large
gene families. Other than to the well characterized cysteine protease 14, another cysteine
protease was isolated (PGSC0003DMP400045977) and also a cysta-tin homologue (a
putative cysteine protease inhibitor PGSC0003DMP400018076). A Kunitz-type proteinase
inhibitor (PGSC 0003DMP400016823) and a subtilase family protein
(PGSC0003DMP400008705) were also identified to be important proteins. It is possible
now to investigate these genes on functional level as now there exact identity is known.

Conclusion:
Phytophthora infestans is a very well-studied pathogen ever since it has caused the
historical Irish famine of Potato. As we entered into the modern era of genomics, so does
the plant pathogen. Although previous efforts for the development of a durably resistant
potato cultivar against P.infestans were not particularly successful but with better
understanding of host-pathogen interaction, at molecular and gene level, will eventually
lead to development of durable resistance. This article is an effort to state brief overview of
important genes or gene products which are proved to play an essential role during
interaction of P.infestans with potato. One crucial gene or a combination of genes can be
selected for further studies of developing resistance against P.infestans.
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