Rebuttal Letter for “Changes in phytohormonal signaling in response to anthracnose in plants.”

Alden Parker – BS 860, Endocrinology

Dear Dr. Zambito,

Thank you very much for your critique of my review article. As per the Journal of Endocrinology’s “instructions for authors,” I’ve highlighted new text in YELLOW, and revised, or changed (i.e. shortened/deleted) text in GREEN. Moreover, in this submission, I have

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adhered to all of their formatting guidelines, including: title page, double spacing, continuous line numbering, and page numbers on the bottom left of each page.

As per your requests, I changed my wording in every area you suggested in the abstract and the article. In sections where my meaning was not clear, I attempted to clarify my points (for example see lines 64 – 65 in this document, which you had marked as “?” on page 2 of my first submission).

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Most importantly, I expanded the JA and SA signaling section to include more information about PAMP/DAMP receptors, the signaling pathways upon receptor activation via PAMP binding, and the JA and SA – mediated responses as a result. I have also trimmed down some of the subsequent hormone sections in order to stay within the word limit.

Overall, I believe this is a much better paper given your critiques and my addendums, so thank you very much for taking the time to thoroughly review my article and to point out areas that could be improved. In writing this review, I really learned a lot about a subject that

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I was veritably ignorant of, and it will absolutely benefit me in my workplace. I would also like to thank you for allowing me to write a more unconventional endocrinology paper on material that we didn’t even come close to covering in the course. I look forward to any and all feedback regarding my final submission.

Best,

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Alden Parker

aldenjacksonparker@gmail.com

423-368-1420

21 S. Wyoming Ave. APT #1

Ardmore, PA 19003

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TITLE PAGE:

Changes in phytohormonal signaling in response to anthracnose in plants6

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BS 860 – Endocrinology

Corresponding Author: Alden Parker: Master’s Candidate, University of the Sciences in Philadelphia

Email: aldenjacksonparker@gmail.com

Postal: 21 S. Wyoming Ave. APT #1, Ardmore, PA 19003

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Keywords: phytohormones, anthracnose, disease resistance, plant immunity,

jasmonic acid signaling, jasmonate, salicylic acid signaling, salicylate

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Word Count:

Abstract: 222

Article: 4,780

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Abstract: Anthracnose is an infection in plants that causes cankers in tissue due to

chronic inflammatory signaling. Its main cause in agricultural plants is Colletotrichum fungi.

Upon challenge, phytohormones initiate innate inflammatory cascades, which respond by

altering the balance of growth, survival, propagation, and immunity. Because the infection

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can be vertically transmitted, often the best way to stop the disease is to kill infected plants,

so it even has the potential to impact conservation of endangered species. Herein, an

examination of anthracnose resistance in plants is described with particular consideration to

alterations in phytohormonal signaling post-infection, and potential supplements to the

immune response are outlined based on recent findings. Anthracnose is detrimental to the

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financial viability of many agricultural entities, so anthracnose resistance has emerged as a

highly sought after trait in agriculture biotechnology. The resistance of Colletotrichum to

fungicides is also examined. Potential genetic therapy targets are proposed to minimize fungi

virulence and harmful inflammatory signaling in order to minimize anthracnose infection in

plants. Finally, exploitation of anthracnose infection for the purpose of enriching therapeutic

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agents for human treatment is proposed. Taken together, the findings presented are used to

craft a strategy that utilizes resistance genes in combination with peripheral supplements to

phytohormonal signaling in response to anthracnose infection in an attempt to maximize

resistance as well as crop quality and yield.

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Introduction: 15

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Anthracnose, Colletotrichum fungi, and Issues in the field: Anthracnose is considered one of

the most severe pathogens to deciduous trees and an array of agricultural species, including: corn (Huffaker et al.,

2011), hot peppers (Lamsal et al., 2012), beans (Campa et al., 2014), rice (De Vleesschauwer et al., 2014), grapes

(Poolsawat et al., 2013) and more. In short, anthracnose is a broadly used term to mean an infectious agent

(bacterial, viral, but most often fungal) that causes tissue damage local to the site of infection. This damage is often

exacerbated by inflammatory signaling from the host innate immune response, which is largely initiated via plant 16

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hormone (phytohormone) signaling (Denancé et al., 2013; Savatin et al., 2014). The concerted effects of the

infection and subsequent inflammation can result in decreased yield, quality issues, growth retardation,

compromised immune responses to other plant pathogens, or responses to stresses, in general, and even the

complete obliteration of entire crop populations (Pakdeevaraporn, 2005). Because anthracnose can be vertically

transmitted due to infection of seeds, often the most effective method of controlling the spread of the disease is to

kill infected plants, which can have a serious impact on the financial viability of many crops, rendering farmers and

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agricultural corporations unable to garner profits as a result of anthracnose, delaying sustainability. Thus, it is

common practice to implement an integrated pest management strategy to prevent anthracnose infection, which

can include: optimized watering, fertilizing, and fungicide spraying conditions (Kim & Yun, 2013), supplementing

the immune response with “inert” plant pathogens, induced overexpression of particular substances, or treatment

with chemicals to confer enhanced resistance (Lamsal et al., 2012; Kim & Yun, 2013; Kim YC et al., 2014;

Planchamp et al., 2014; Seo et al., 2014; Yun, 2014; Narusaka et al., 2015). While this is an improvement compared

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to no pest management, it does not address problems that arise with anthracnose infection. Hence, there exists a

need to optimize anthracnose resistance in crop plants.

Anthracnose in crop plants is most often caused by fungal infection of the Colletotrichum genus (Bailey & Jeger,

1992), with multiple strains being able to infect the same host or multiple hosts (Than et al., 2008), resulting in a

variety of individual immune response mechanisms (Campa et al., 2014). Resistance genes to anthracnose have

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been found to be monogenic or multigenic (Pakdeevaraporn, 2005). Therefore, it is important to identify

resistance genes against all potential anthracnose agents in a desired crop plant to allow for an optimal breeding

strategy for maximal resistance. Unfortunately, this is an arduous process with significant costs but has the

potential to pay off should a high level of resistance be achieved. Thus, it is worth the expenditure of time and

resources to develop these assays before planting a crop for production.

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Interestingly, Colletotrichum express mutualistic lifestyles depending on the particular genotype of the host they

infect, and are thus termed “endophytes.” In this manner, the fungi live symbiotically within the host plant,

oftentimes providing enhanced resistance to peripheral environmental stresses such as drought and herbivores

(Rodriguez & Redman, 2007; Cheplick & Faeth, 2009). It can also be said these parasites are “biotrophic” as

opposed to “necrotrophic” in that they don’t kill their host and then feed on dying plant tissue. In biotrophic cases,

determining the genetic basis for the observed symbiosis could provide a platform for which to design breeding

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strategies to maximize anthracnose resistance. To that end, many resistance genes have been identified in

different species (Pakdeevaraporn, 2005; Narina et al., 2011; Yang H et al., 2012). Hence, via integrated pest

management field studies combined with genome-wide association studies (GWAS), quantitative trait loci (QTLs)

can be identified for effective breeding strategies for anthracnose resistance, or in some cases, symbiosis, should

the latter be the more viable option for fruit or seed production.

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One of the major problems with integrated pest management strategies is that many fungi strains have developed

a high level of resistance to fungicides (Kim S et al., 2014). Therefore, it is worthwhile to examine fungicide

resistance genes within pathogenic fungi to determine if there are better alternatives to the particular fungicide

theoretically being used in pest management. If a problematic fungus has a resistance gene that is not targeted or

accessible by the planned fungicide, then it will remain problematic. Doing this in combination with identifying

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fungal-resistance genes in plants will provide for an optimal breeding scenario because resistance in both plants

and fungi are considered.

Arabidopsis thaliana is widely used because of its short life cycle, high number of progeny, and small requirement

for growth space (Koornneef & Meinke, 2010). Thus, it is an advantageous model organism. However, it is a

dicotyledon, so it inherently has limited applicability to monocotyledons like maize, wheat, and rice, all of which

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are socio-economic food staples worldwide. Thus, there exists a need to develop other model plants to increase

the comprehensiveness to plants with higher homology to the model plant and to plants where the socio-

economic impact of crop loss is significant. The most obvious monocot that comes to mind would be rice because

of similar factors to why Arabidopsis is such a good model, but there are likely more efficient grass species that

would save on time and money in their growing and maintenance for resistance trials.

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Finally, many plants contain biosynthetic compounds that have the potential for therapeutic use in the treatment

of variety human diseases. One of the most famous is aspirin, which is a component of willow tree bark extract

(Wick 2012). It is worth noting that new natural compounds are discovered virtually every day. Should these

compounds have therapeutic potential, the source of the compound instantly becomes a hot commodity. That

said, the source has the potential to be overused, such as the overharvesting of the Pacific yew tree in the case of

the cancer treatment drug taxol, prompting the medical and chemical industry to discover alternative sources of

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the drug, including: synthesis, semi-sythesis, and cloning-based techniques (Nicolaou et al., 1994). These

techniques often prove costly to develop and difficult to produce a high yield of comparable quality product to the

true biosynthetic compounds, so it often takes years and even decades before a cost-efficient method of high-

quality synthesis is achieved. To counteract the research and development of synthesizing these nature-derived

drugs, a rapid propagation technique is often employed to produce a relatively large number of offspring of the

compound source in a short amount of time. Therefore, if the source plant is susceptible to anthracnose, then it is

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of utmost importance to include the resistance considerations previously described when formulating a breeding

strategy for large scale therapeutic production of naturally-derived treatments.

Phytohormones and Immune Response Initiation:

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Many of the adverse effects resulting from anthracnose infection are due to the inflammatory signaling of the host

innate immune response, which is primarily driven by phytohormonal signaling (Denancé et al., 2013; Savatin et

al., 2014). Moreover, there is a disruption in the balance between growth and immune signaling as a result of

infection, often to the detriment of the plant (Denancé et al., 2013). Hence, it is of particular interest to determine

how expression of particular phytohormones is altered after anthracnose challenge, and insight into expression at

local infection sites may elicit a greater understanding of systemic resistance (Vlot et al., 2008; Fu & Dong, 2013).

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Jasmonic Acid (JA) and Salicylic Acid (SA): Jasmonic acid is involved in light responses and

especially in the production of defense proteins (Antico et al., 2012). JA signaling is hypothesized to be the front

line of defense against necrotrophic pathogens, especially in combination with ethylene (De Vleeschauwer et al.,

2014). There appears to be a high level of crosstalk between JA and Salicylic acid that is highly conserved across a

variety of plant species, and the signaling of each is dependent upon the type of pathogen encountered(De

Vleeschauwer et al., 2014). Both JA and SA have been proposed as signals for systemic acquired resistance (Fu & 30

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Dong, 2013). SA is hypothesized to be the primary defense against biotrophic pathogens (De Vleeschauwer et al.,

2014). Thus, plant immunity appears to be a tailored response dependent upon the type of pathogen. Pathogens

express certain chemical markers, pathogen-associated molecular patterns (PAMPs), and induce changes in plant

tissue that the plant recognizes as dangerous to self, or damage-associated molecular patterns (DAMPs).

Therefore, it appears that JA signaling is more crucial to DAMP responses, with SA signaling be the major response

pathway to PAMPs (Denancé et al., 2013). This corresponds to the hypothesis that JA signaling is upregulated for

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necrotrophic pathogens, and SA signaling is upregulated for biotrophic pathogens. For hemibiotrophic pathogens,

those that begin as biotrophic and develop into necrotrophic, it is likely a combination of JA and SA signaling,

depending on the stage and consequence of infection. It is likely that SA signaling dominates early on in infection,

with a switch in the balance to JA signaling later in infection when tissue starts to become damaged. However, it is

not always as simple as it seems to be.

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PAMP receptors are known as pathogen recognition receptors (PRRs). PRRs in plants are much like PRRs in

mammals in that they are primarily protein structures that have an endodomain, ectodomain, and a

transmembrane section that spans both the cell membrane and cell wall (Macho & Zipfel, 2014). Upon PAMP

binding, immediate responses are an increase in ion fluctuation, an oxidative burst, and an activation of mitogen-

associated protein kinases (MAPKs), resulting in an increase in protein phosphorylation (Boller & Felix, 2009). Early

responses are ethylene biosynthesis, PRR endocytosis, and gene activation to increase production of PRRs and

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defense molecules like SA and JA, depending on the pathogen (Boller & Felix, 2009). Late responses are to inhibit

seedling and whole plant growth in a sequestration of resources to fight off the pathogen (Boller & Felix, 2009).

Upon the increase in JA synthesis, jasmonate ZIM-domain proteins (JAZ), which repress JA signaling under

normal conditions, become ubiquitinated for degradation (Wager & Browse, 2012). This results in the release of a

MYC2 transcription factor that binds to a nuclear receptor, coronatine-insensitive protein 1 (COI1), and drives

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production of abscisic acid (ABA), a growth retardant (Smith et al., 2009) and also increases the responses

associated with PRR activation described above (Vasyukova & Ozeretskovskaya, 2009). Mutation of COI1 results in

severe susceptibility to necrototrophic pathogens (Smith et al., 2009).

SA upregulation largely results in cell death at the site of infection but interestingly confers cell survival in

plant tissue distal to the infection site (Gimenez-Ibanez & Solano, 2013). Under normal circumstances, SA levels are

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low, and the SA receptor NPR-1 localizes in the cytoplasm by oligomerizing to itself, but upon SA increase,

oxidative stress in the cytosol causes the NPR-1 to monomerize and enter the nucleus where it binds to TGA clade

transcription factors, producing more SA and PRR-activation associated responses (Gimenez-Ibanez & Solano,

2013). Mutation of NPR-1 results in severe susceptibility to biotrophic pathogens (Gimenez-Ibanez & Solano,

2013).

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Abscisic Acid (ABA): Abscisic acid is one of the most significant stress and growth regulation plant

hormones. As the balance between growth and stress signaling is disrupted from pathogen infection, ABA

signaling increases with biotic stress, which results in growth retardation (Cutler et al., 2010). ABA was shown to

inhibit pathogen entry through the stomata, promoting resistance, but has also been shown to interfere with other

response pathways (Hwang et al., 2009), decreasing resistance depending on the pathogen type, time of infection

during plant development, and tissue site of infection (Ton et al., 2009). Thus, the true immune function of ABA 37

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remains largely unclear because of circumstantial contradictions, but it is clear that its expression level is somehow

involved in the global immune response. Maize root inoculation with rhizobacteria resulted in systemic resistance

to anthracnose infection, which was mediated by a combination of JA and ABA signaling early in development

(Planchamp et al., 2015). This suggests that ABA may play a larger role in innate immunity when the plant is in its

main growth stage, before it has begun to produce fruit in an attempt to propagate itself.

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Auxins: Auxins have multifaceted effects in plants, including: cell enlargement, root and flower

initiation, growth regulation in conjunction with other phytohormones, and regulation of expression of other

phytohormones (Zhao, 2010). Similar to ABA, auxins have been shown to have positive or negative effects in

regard to disease resistance (Denancé et al., 2013), and again, it is largely dependent on the pathogen type, time of

infection during development, site of infection, and how other phytohormones are expressed post- infection. It

was shown that an increase of microRNA that targets auxin receptors yields plants that are more resistant to 39

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biotrophic infection but less resistant to necrotrophic infection, and a balance between auxin and SA signaling in

response to infection was proposed (Robert-Seilaniantz et al., 2011). Moreover, SA was shown to inhibit pathogen

growth in Arabidopsis by repressing auxin signaling (Wang et al., 2007). Thus, auxin signaling appears to be more

suppressive in pathogen resistance, but its true function is elusive. Given the fact that auxin signaling is highly

involved in cell enlargement, it makes sense that high auxin signaling would allow for easier pathogen entry as the

cell wall loosens. There have not been many studies regarding auxin signaling in response to anthracnose, but

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since it clearly interacts with expression levels of other immune response hormones and is involved in regulating

the balance between growth and stress, it will be an important system to analyze moving forward.

Cytokinins (CKs): Cytokinins are involved in shoot development and mitosis and are also peripheral

growth regulators, as they are highly concentrated in the shoot apical meristem (Hwang et al., 2012). They are also

highly interactive with auxin signaling in shoot formation during early embryogenesis, but in contrast to auxins,

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there is evidence that they suppress immune response to biotrophic pathogens (Walters & McRoberts, 2006),

which anthracnose causing agents sometimes are. Some pathogens secrete cytokinins of their own to disrupt

normal signaling (Hwang et al., 2012), most likely to create a more favorable environment for their own

reproduction. Alterations in CK levels after anthracnose infection have been determined in maize (Behr et al.,

2012), indicating that CK signaling is important for both proper host growth and immune response after

necrototrophic pathogen challenge, but the exact role CK signaling plays has yet to be discovered. Thus, CKs

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appear to suppress immune signaling in response to biotrophic pathogens while they activate immune response

signaling in response to necrototrophic pathogens, but more studies involving anthracnose are required.

Ethylene: Ethylene is involved in cell growth and shape, and in turn, growth modification in response to

stress. It is highly interactive with other signaling pathways (Yoo et al., 2009). Its role in growth is apparent from

visualizing the hyponastic response, in which submerged plants project long leaves and stems through water to

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obtain normal air exchange above the surface, where ethylene is soluble (Polko et al., 2013). Thus, an excess in

ethylene will cause excess growth in an attempt to regain proper balance. Auxins, CK, and stress have been shown

to promote ethylene synthesis, while JA, ABA, and Auxins promote ethylene signaling (Yoo et al., 2009). Ethylene

and JA-dependent defense signaling has been identified in wheat (Gottwald et al., 2012) and kimchi cabbage (Lee

et al., 2014), and plant immune responses are largely thought to be centrally dependent on ethylene, JA, or SA

signaling, or a combination of them depending on the pathogen (Denancé et al., 2013). Supporting the case for

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ethylene with JA, its biosynthesis pathway was shown to be upregulated upon PAMP recognition (Spanu et al.,

1994). In support of ethylene and SA, ethylene was shown to have two spikes in production in tobacco, a dicot,

after Colletotrichum challenge: one about 24 hours before the necrotrophic phase when cankers began forming,

and another late in the necrotrophic phase when the cankers began to expand (Chen et al., 2003). Thus, there is

evidence that ethylene is produced in direct response to pathogen entry and tissue damage in response to all

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pathogens. In general JA and ethylene seem to work in a concerted manner (Gottwald et al., 2012; Lee et al.,

2014), but the relationship between ethylene and SA appears to be more complex and circumstantial.

Gibberellins: Another class of phytohormones newly-found to be involved in JA and SA-dependent

immune signaling is gibberellins (De Bruyne et al., 2014). Gibberellins initiate growth by degrading a group of

growth suppressors called DELLA proteins (De Vleeschauwer et al., 2014). They are mostly involved with seed

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germination and early development, but have recently been implicated as players in JA and SA signaling activation

or repression, possibly by their DELLA protein degradation, as DELLA proteins have been implicated in disrupting

the JA/SA signaling balance (Navarro et al., 2008). There appears to be significant crosstalk between JA and

gibberellins, as JA has been shown to increase immunity by inhibiting gibberellins (Yang D et al., 2012). Thus,

gibberellins appear to be inhibitory to plant immunity, largely due to their high level of involvement in driving

growth. Interestingly, gibberellins have been implicated in inhibiting ethylene synthesis and are agonistic to its

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signaling (Yoo et al., 2009), indicating that they may have a more peripheral effect on immune signaling.

Altogether, gibberellins appear to be involved in immunity tangentially through their high involvement in growth

regulation. Thus, once the balance of growth and stress is disrupted upon infection, gibberellin activity is

decreased, allowing DELLA proteins to inhibit growth and affect the JA/SA signaling balance accordingly to the

given pathogen.

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Other phytohormones: The hormones previously mentioned are some of the major effector families

in phytohormonal immunity signaling. Others include brassinosteroids, which are involved in general stress

response (De Vleeschauwer et al., 2014), nitric oxide, which is involved in seed germination and general defense

(Baudouin & Hancock, 2013), and plant peptide hormones, which are primarily involved in intercellular signaling in

growth, development, immunity, and more (Lindsey, 2001; Huffaker et al., 2011).

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Regulation & Crosstalk: Obviously, there are many signaling pathways involved in plant immunity,

and they affect each other based on growth, development, survival, fitness, abiotic and biotic stresses, and

reproduction. In the case of anthracnose, the plant must balance fitness with growth and normal signaling

pathways become altered and affect other pathways. In regard to disease resistance, phytohormonal pathways

have the ability to modulate others, with synergism versus antagonism and positive and negative feedback often

coming into play (Derksen et al., 2013). JA and SA signaling have been hypothesized to counteract each other, in 50

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general (Navarro et al., 2008; Denancé et al., 2013; Derksen et al., 2013, De Vleeschauwer et al., 2014), but one

does not always affect the other in defense signaling (Huffaker et al., 2011). The antagonistic nature of their

interaction would suggest that activation of one pathway would render a plant more susceptible to infection from

a pathogen that would normally be combated by the other pathway. However, they have also been shown to have

a concerted effect in particular cultivars for anthracnose infection (Lee et al., 2014), which makes sense in the case

of hemibiotrophic pathogens. Thus, the relationship between JA and SA signaling is complex and largely depends

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on the pathophysiology of the given pathogen. Since there are a variety of causes of anthracnose inducing a variety

of immune responses, a ubiquitous generalization is impossible to make regarding JA/SA signaling.

Outside of JA/SA regulation, other phytohormones have significant interaction in both normal function

and defense signaling. For example, ethylene is required for auxin-regulated root elongation (Swarup et al., 2007).

As for positive feedback, treatment of corn with a defense peptide results in systemic production of more JA,

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ethylene, and other defense molecules (Huffaker et al., 2011), and defensin overexpression was shown to enhance

anthracnose resistance by upregulating JA signaling in peppers (Seo et al., 2014). Activation and repression of

signaling pathways is largely driven by nitric oxide signaling, especially in the recognition of DAMPs (Baudouin &

Hancock, 2013). ABA and JA have been shown to antagonize each other, lending further credence to the

hypothesis that ABA contributes to susceptibility to necrotrophic pathogens (Derksen et al., 2013). It makes sense

that ABA and JA would be antagonistic because while healthy, a plant would not want to activate a defense

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pathway because it would expend resources that would better be used on growth and reproduction. Thus, there is

a myriad of phytohormonal interplay that is dependent upon environmental and biological stresses, and

interactions of different pathways should be carefully considered when examining phytohormone-driven defense

signaling.

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Supplementing the Immune Response:

Interestingly, many pathogens have developed the ability to mimic plant hormonal signaling (McSteen &

Zhao, 2008; Hwang et al., 2012; Denancé et al., 2013). For example, gibberellins were first isolated from pathogens

(McSteen & Zhao, 2008). Thus, the pathogen would ideally disrupt normal growth and development signals and

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induce stress signals for the benefit of its own propagation. This contributes to the complexity of studying

phytohormone-driven immune signaling and highlights the ability of foreign molecules to affect the growth/stress

balance and systemic immune response. While this is a strategy that is advantageous for pathogens, it also allows

for supplementation of the immune response with foreign agents to confer disease resistance. Plants have been

shown to have a sensing system for foreign pathogens (Hiruma et al., 2010), much like cognate antigen

presentation in human cells. This, too, can be employed to supplement the immune response, similar to

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vaccination against disease. These are aspects that should be considered in developing an integrated pest

management strategy (Kim & Yun, 2013).

Treating with or overexpressing native plant molecules to induce defense signaling has been studied

(Huffaker et al., 2011; Seo et al., 2014), and β-Amino-n-butyric acid (BABA) increases anthracnose resistance (Kim

YC et al., 2014), but foreign substances can also be used as immune supplements. Antibiotics from rhizobacteria

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application have anti-fungal properties in hot peppers (Lamsal et al., 2012), which is a case of infecting a plant with

a beneficial, inert pathogen to obtain resistance against a virulent one. Similarly, rhizobacteria application also

been shown to increase anthracnose resistance in corn (Planchamp et al., 2015), and myxobacteria application has

shown similar results in peppers (Yun, 2014). Even fungal agents can be used. Yeast cell wall application was shown

to induce phytohormonal defense responses against anthracnose infection by activating JA signaling early and SA

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signaling late in the infection cycle (Narusaka et al., 2015). There is a long history of symbiosis upon which both the

plant and fungus are dependent (Rodriguez & Redman, 2007).

It should be noted that application of foreign substances to supplement plant immunity sounds like a

novel idea that could decrease morbidity, but the effect it has on disrupting normal growth should be accounted

for in breeding strategy and in quality and yield. Whether or not these foreign substances end up in edible fruit of

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the plant should also be monitored at the risk of introducing potential carcinogens into food. At the very least, it

has the potential to reduce vertical transmission, allowing for an improvement in progeny. Thus, it merits further

investigation.

Sources of Resistance, Fungal and Agricultural:

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Many fungal strains have proven resistant to fungicide application, based on their defensive ATP-binding

cassette (ABC) transporters (Espinel-Ingroff, 2008). The particular transporter was successfully identified in

Colletotrichum acutatum, which causes anthracnose in hot peppers (Kim S, 2014). Hence, ABC transporters can be

potential fungal targets for inhibition via their structural determination and subsequent rational drug design.

Gaining more insight into the evolution and mechanisms of fungicide resistance will provide a platform for which

to prepare for resistance development. Also, deep RNA sequencing of Colletotrichum gramnicola, which causes

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anthracnose in corn, revealed alternative splicing mechanisms for characterization of gene models (Schleibner et

al., 2014). Thus, there is a basis for which to compare genetic homology across fungal species to identify resistance

genes and expressed molecular targets for fungicide development or drug inhibition in the case of mimicking

molecules. Moreover, profiling the phytohormonal immune responses to different pathogen strains can provide

insight in how best to combat them (Campa et al., 2014).

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As there are resistance genes in fungi, so there are resistance genes in plants. Particular cultivars have

been proven to be more resistant to anthracnose, and it is likely due to a genetic basis (Narina et al., 2011; Lee et

al., 2014). Hence, particular resistance genes can be identified via knockout studies and monitoring phytohormonal

signaling as a result. With the genetic basis identified, other species can be screened for homologous resistance

genes, or the resistance genes can be cloned into other species to create highly resistant transgenic plants. Novel

techniques like Next Generation Sequencing (NGS) have also been extensively applied for tagging resistance

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markers (Yang et al., 2012). Thus, progeny of unknown genotype can be analyzed with seed chip DNA to determine

their resistance profile, saving time and money. Once particular resistance genes are identified, they can be

overexpressed via agrobacteria transformation to confer increased fungal resistance, and this can even be done

across species to induce transgenic resistance (Singh et al., 2015). Clearly, doing the research on both plants and

fungi before diving into the deep end of high-scale field production is a worthwhile venture to the agricultural

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biotechnology industry and should be included when developing integrated pest management strategies to

minimize crop loss and cost while maximizing quality, yield, and pathogen resistance.

Conclusion and Future Directions:

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Monitoring phytohormonal responses to anthracnose-causing pathogens is a viable method of

determining how best to combat the given pathogen. Should a resistant cultivar be identified, it can be propagated

in a controlled environment to prevent vertical transmission, mutagenized and propagated again to produce

mutant progeny. Then the progeny can be challenged with infection, followed by phenotypic, genetic and

phytohormonal analysis of mutants with altered resistance to identify the genetic basis of resistance. The future of

anthracnose resistance lies within reverse genetics studies like these combined with genome-wide association

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studies between cultivars to determine what the optimal hybrid genome is. This in combination with identification

of fungicide resistance genes in fungi strains will allow for an optimal scenario for financial viability of crop

production.

Rice is emerging as a tractable model for plant studies (De Vleesschauwer et al., 2014), so there is at last a

monocotyledon model for which to compare other crop plants and their resistance profiles to anthracnose

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pathogens. That is not to say that Arabidopsis is no longer useful, but that findings using it as a plant model should

be confirmed in rice before applying generalizations regarding phytohormone-driven defense signaling. The future

of developing anthracnose-resistant crops depends on the comprehensive development of a highly homologous

and applicable, relevant model organism with which to conduct studies. While rice is a good start, it is likely that

there are even more advantageous grass types in terms of growth requirements and cost, so grasses with short life

cycles should be examined as a more applicable model organism for comparison to dicotyledons.

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This is not only applicable to the agriculture biotechnology industry, but it is also pertinent to the medical

world. Many plants that are endangered are so because of their high susceptibility to a given pathogen. If they are

used by humans or animals for some sort of medicinal purpose, this only exacerbates their population issues.

Therefore, integrated pest management combined with propagation and breeding strategies can be implemented

with pathogen resistance in mind. Anthracnose does not always have to be an enemy, either. In terms of

pharmacognosy, anthracnose and other pathogens have the potential to be used to induce an increase in

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production in stress hormones, which can be used to treat human cancer and other diseases (Fingrut & Flesher,

2002). Moreover, plants under stress produce increase amounts of other non-hormonal therapeutic compounds.

This makes it easier and cheaper to produce hard-to-synthesize natural therapeutics while reducing the impact on

the population source by producing more of the compound from fewer source plants. At this point, such a

technique is merely a theory, but it does have the possibility of being a viable technique. Only through

phytohormonal monitoring post-infection will it be determined if anthracnose infection could be used to induce a

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higher production of a therapeutic substance. It will also provide greater insight into initiation of innate immunity

in plants, which even though it is a much older pathway than in humans, it is less understood. Given the fact that

the human population is growing, food availability will become a significant issue for much more of the world in

the near future. Thus, determining how to optimally grow grain foods that are highly resistant to stresses of any

kind must be common practice in the future of agriculture.

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