Evolution of pathogenicity in obligate fungal pathogens and allied genera

Evolution of pathogenicity in obligate pathogens

A characteristic feature of obligate pathogens is the ability to inhibit recognition by the host and suppress host defense mechanisms. This characteristic feature developed independently in distantly related clades of fungi and oomycetes (Thines & Kamoun, 2010; Kemen & Jones, 2012). Characteristic features like host entry and haustoria formation are brought about through similar changes within the genome and the proteome. Another characteristic is a lack of lytic enzymes found in obligate biotrophic filamentous fungi and oomycetes (Kemen, Agler & Kemen, 2015). Similar characteristics could be the result of identical genetic changes caused by mutations that are identical in independent decedents from a common ancestor (parallel evolution) or by introducing an allele from a line into another related line by hybridization (Stern, 2013) or horizontal gene transfer (HGT) (collateral evolution) (Fig. 5). With the availability of genomic sequences, it may be argued that all of them contribute to observed convergence. Characteristic biotrophic adaptations of obligate parasites for existence inside plant cells are as follows.

Collateral evolution

HGT is an example of collateral evolution, and it causes a transfer of genes from fungi to oomycete genomes. Although the exact mechanism by which this HGT occurs is unclear, HGT has been demonstrated to be an important part of collateral evolution between fungi and oomycetes (Savory, Leonard & Richards, 2015). In obligate pathogens, the genes transferred encode proteins that attack or feed on plants (Richards et al., 2011). Hemibiotrophic oomycetes are more likely to exhibit HGT than obligate biotrophic oomycetes such as H. arabidopsidis and Albugo laibachii. H. arabidopsidis has been reported to have 21 putative HGTs (representing 3.6% of the secretome); computational validation was possible with only one HGT in Albugo (Richards et al., 2011). The HGTs transferred from fungi to oomycetes encode for proteins that assist in sugar degradation, transportation, and reorganization. HGTs from fungi to oomycetes involve genes coding for proteins that function as catalysts in the metabolism, transport, and structural changes in sugars. However, obligate biotrophs do not possess many plant degrading enzymes to avoid defense activation. Because lytic enzymes are harmful for obligate pathogen sustenance, it will not be in the best interest of the pathogen if these genes are transferred. Effectors with no known function and origin are race-specific and usually are not functional in other organisms.

Oomycetes that possess diverse families of Nep1-like proteins (NLPs) which are species-specific have undergone one HGT event and experienced considerable divergent selection (Soanes & Richards, 2014). As well as causing necrosis in certain host species, NLPs can contribute to virulence and disease (Gijzen & Nürnberger, 2006). A comparison between downy mildews and Phytophthora revealed NLPs are reduced in downy mildews, with regards to Phytophthora (Baxter et al., 2010). The 12 NLP-coding genes found in downy mildew are not responsible for necrosis (Cabral et al., 2012). In contrast to its hemibiotrophic relative, the biotrophic pathogen’s proteins appear to have evolved over time (Baxter et al., 2010). This also suggests a possible role in microbe–microbe interaction. In gnotobiotic systems, the function of NLPs as environmental factors can be determined by using downy mildew NLP-knockout mutants. By HGT, a virulence factor (e.g., NLP) introduced between populations can give rise to identical phenotypes (collateral evolution), however over time the development of these new functions may cause confusion regarding the original similarity (Fig. 5).

Host jumps to escape environmental pressure

Host jumps are essential for pathogen survival in a host. The phenomenon is brought about by effector molecules which allow for infection of and survival within another host. This is seen when the phylogenetic distance is short among the two hosts with similar effector targets. This can also be seen in the hemibiotrophic lineage Proteus mirabilis (Dong et al., 2014).

A drastic change, such as gene loss, is required if the jump is made from a monocot plant host to a dicot plant host as in the case of Melanopsichium pennsylvanicum (Sharma et al., 2014). A bigger jump to a host that is very distantly related happens when the defense mechanism of the new host is suppressed in relation to the original host. When susceptibility does not occur, natural infection can be observed in a nontraditional host. This occurs when the pathogen silences its own defense mechanism (Cooper et al., 2008). Host jumps happens when the immunity of the host is in question and when induced susceptibility is a natural phenomenon. An example is spore dispersal to a dead nearby host (Kemen, Agler & Kemen, 2015). This is an example of an environmental change that leads to temporary infection abilities which help in host jumps by the pathogen (Antia et al., 2003).

Compatibility of pathogens leads to susceptibility of the isolates that were not compatible at first (Ouchi, Oku & Hibino, 1976; Heath, 1980). Several studies have confirmed this in rust and powdery mildew fungi. If plants are co-infected, their susceptibility is limited to a few cells away from the primary infection site. Under such conditions, reproduction may not be possible (Kemen, Agler & Kemen, 2015). The induced susceptibility of oomycetes between A. candida and H. arabidopsidis has been studied as well. A.candida and H. arabidopsidis co-infect Brassica sp. naturally. When appropriately pre-infected, A. candida greatly enhanced the disease-causing ability of compatible H. arabidopsidis, but Albugo showed a lower multiplication rate (Singh et al., 2002). An infection with a non-sporulating H. Arabidopsidis was caused by rapid spore formation caused by virulent A. candida (Kaur et al., 2011). Isolates of the same species that are incompatible cannot cause susceptibility to A. candida (Singh et al., 1999). Thus, susceptibility that is induced and not natural is efficient among microbes that utilize the same target effectors or other resources. There has also been evolution to limit resource competition among obligate biotrophs, perhaps through effector mediated relationships.

Role of transposable elements (TEs)

TEs have a role in evolutionary changes that lead to pathogenicity and survival ability (Manning et al., 2013). They are also responsible for gain and loss of gene function, chromosomal rearrangements, and complete inactivation of genes (Biémont, 2010). Expansion in genome size, alternative splicing and exonization, alteration of gene expression, alteration of a regulatory network, epigenetic control, and TEs all contribute to genome plasticity. Genome plasticity enables organisms to adapt to environmental changes. Adaptive evolution mediated by TEs is facilitated by recombination events resulting in genomic diversification. This is achieved through genomic changes which persist under positive selection in obligate fungal pathogens.

TEs induce pathogenicity by their proximity with avirulence/pathogenicity associated genes. TEs are known to gain virulence through deletion of avirulence genes, and they promote pathogenicity by inducing nucleotide diversity. Mutations from TE insertions can lead to genetic variability that generates many new pathogenic variants with conferred ability to invade previously resistant host plants (overcome host plant resistance) and hence expand on the host range. TEs are known to alter host fitness through deleterious insertions, driving speciation and adaptive evolution.

The occurrence of many TEs in wheat biotrophs had led to a rapid evolution of the genome (Amselem, Lebrun & Quesneville, 2015). As an example, we have seen an expansion of the genome size of rust fungi, often between 100 and 200 Mb (Cuomo et al., 2017). Most genome sizes of other basidiomycete fungi are less than 50 Mb (Duplessis, Bakkeren & Hamelin, 2014). The larger size is mainly contributed to from high amounts of repetitive DNA referred to as TEs. A Pst race PST-130 has TEs which contribute to 17.8% of the genome. The genome of the Chinese isolate of CYR32 has 50% TEs, hence the genome size almost twice as large (Zheng et al., 2013; Cuomo et al., 2017). A member of the Puccinia genus, Pucciniatriticina (Pt), had a higher repeat content, with an average of more than 51% (Cuomo et al., 2017). The Bgt genome had even more TEs, reaching 90% (Wicker et al., 2013).

Role of secreted proteins

Obligate biotrophic pathogens are associated with secreted proteins (SPs) that help with escaping host immune responses (Fig. 4). Species-specific genes coding for SPs were found in the genomes of rust and powdery mildew. The Pst genome contains 2092 SPs, which account for 8.3% of the total number of predicted protein genes (Zheng et al., 2013). Two Pt races produced 660 SPs (Kiran et al., 2016), while a member of the Puccinia genus, Pgraminis f.sp. tritici (Pgt), produces 1459 SPs (Zheng et al., 2013).

A virulent Pt isolate showed more SPs than race-specific isolates with a narrow virulence scope. These SPs are unique to each species of the pathogen because of the fast-evolutionary modification of the protein. The rapid evolution of some effectors indicates their individual pathogen specificity. As discussed previously, of the three rust fungi, 62% of SPs are unique to that species. It has been shown that 5% of SPs are found only exclusively in the rust and powdery mildew fungi (Spanu et al., 2010; Dean et al., 2005), indicating that each pathogen evolved differently.

Role of haustoria

Haustoria (Fig. 1B) are associated with effector delivery and nutrient uptake (Voegele et al., 2001). RNA transcriptomic studies of Pgt haustoria and germinated urediniospores showed genes that were upregulated in germinated urediospores. These genes were associated with cell proliferation, cell wall synthesis (Upadhyaya et al., 2014), and DNA replication. The haustorium is crucial for biotrophic colonization of Pst as it enhances expression of genes involved in ATP and TCA synthesis (Garnica et al., 2013). A total of 520 secreted proteins (HSPs), 430 upregulated secreted proteins in haustoria, and 90 genes were identified for Pgt (Cuomo et al., 2017). To identify specific avirulence alleles whose transcripts bind resistance genes, the effectors must interact with their corresponding targets (Flor, 1959; Moseman, 1959). Rust and powdery mildew produce effectors in haustoria, which are then transferred to host cells. Novel effector haustorial proteins are described here (Elmore et al., 2020; Garnica et al., 2014; Link et al., 2014; Lorrain et al., 2019; Polonio et al., 2019; Tao et al., 2017). U. fabae’s haustorium expressed the rust transferred protein 1 (RTP1), which translocated into the cytoplasm of the host during the interaction (Lawrence, Dodds & Ellis, 2010). Stripe Rust CYR31 race haustoriums contained 1,197 secreted proteins, 69 of which inhibited tobacco cell death and 49 of which suppressed wheat callose deposition. Transcriptomic studies were used to identify these proteins. Infection processes are associated with these proteins in P. striiformis (Xu et al., 2020). It is possible to further screen these effector proteins identified by haustorial studies for features associated with avirulence.

Role of effectors

There have been no definitive studies determining whether rust fungi effectors play a role in pathogenicity. In rust pathogens, there were no knockout mutants available. Small interfering RNAs (siRNAs) obtained from pathogen dsRNA can be expressed in plants. These siRNAs were able to enter the pathogen and silence their transcripts (Jaswal et al., 2020), which is referred to as host-induced gene silencing (HIGS). Fungi, oomycetes, and insects have been shown to exhibit this phenomenon. Based on this information, a Barley stripe mosaic virus (BSMV)-mediated HIGS system was created to silence any Pst genes, by expressing dsRNAs derived from Puccinia (Yin, Jurgenson & Hulbert, 2011). It has been demonstrated that BSMV-HIGS inhibited the expression of haustoria-specific genes. In plants, compromising Pst infection also led to silencing effectors (PEC6 and PSTha5a23) (Yin, Jurgenson & Hulbert, 2011; Cheng et al., 2017). This model system helps evaluate rust pathogen effectors via transient silencing.

The rust fungal pathogens are known to contain eight effector proteins, including RTp1 which was transferred from rust pathogens and obtained from Uromyces fabae, four effector proteins from M. lini, AvrP4, AvrM, AvrL567 and AvrP123, and three effectors PGTAUSPE-10-1, Avr35 and Avr50 from P. graminis (Cheng et al., 2017; Petre, Joly & Duplessis, 2014; Prasad et al., 2019; Salcedo et al., 2017). Two additional AVRs were seen in P. graminis: Avr35 and Avr50. P. graminis mutants and non-mutant isolates were analyzed using comparative genetics in order to identify AVRSr35 associates with Sr35 (wheat resistance gene). A mobile element inserted into the AvrSr35 gene altered functional characteristics which also included susceptibility (Salcedo et al., 2017). Another study was able to demonstrate the interaction between the avirulence gene-encoding protein AvrSr50 secreted by haustoria cells and the immune receptor Sr50. AvrSr50 originated from a naturally occurring mutant of P. graminis with a 2.5 megabase pair deletion in its genome. As a result of these groundbreaking studies, susceptibility factors of pathogens have been identified (Cheng et al., 2017). Genome-wide association studies (GWAS) and map-based cloning have identified nonvirulent genes that have structural resemblance to RNase-like proteins seen in wheat and rye powdery mildew pathogens (Praz et al., 2017).

Studies on effector molecules in powdery mildew detected an association between (Mla) genes and barley mildew resistance. These genes have the functional capability to identify effectors from a wide variety of gene families (Jaswal et al., 2020).

Role of small RNA

It is believed that small noncoding RNA molecules, such as microRNA, regulate vital functions in the host (Dubey et al., 2019; Kusch et al., 2018; Mueth, Ramachandran & Hulbert, 2015; Wang et al., 2017a; Weiberg & Jin, 2015; Jaswal et al., 2020). They regulate genes associated with defense mechanisms and immunity at different developmental stages with a functional role similar to effector proteins. It has been shown that sRNAs can move in the cytoplasm and silence the host defense genes of P. striiformis. This movement happens because of the presence of extracellular vesicles (Wang et al., 2017a; Wang et al., 2017b; Jaswal et al., 2020). Genome-wide sRNA association studies in the host and pathogen indicated that sRNA may inhibit the host’s own effector genes and target any genes associated with immunity, such as RLKs (receptor-like kinase) and NBS-LRR (nucleotide binding site- leucine-rich repeat) proteins. As a result, they can interact with the host similarly to how effector molecules do (Dubey et al., 2019; Sperschneider et al., 2018; Jaswal et al., 2020). The presence of these molecules in wheat leaf rust is seen at different developmental stages (resting spores, germinated spores at 16 and 24 h, and highly infected wheat leaf) demonstrated the presence of multiple defense-related genes, like reactive oxygen species (ROS), transcription factors (RLKs), and any resistant genes associated with diseases (Dubey et al., 2019). In P. striiformis (Pstr) there are a wide range of sRNAs that silence any endogenous genes in the host and genes related to defense and immunity (Mueth, Ramachandran & Hulbert, 2015).

Role of secondary metabolites

The secondary metabolites (SMs) act as non-proteinaceous effectors that manipulate the host with toxins (Castro-Moretti et al., 2020; Collemare, O’Connell & Lebrun, 2019; Pusztahelyi, Holb & Pocsi, 2015). SMs also function as nonvirulent factors and suppressing host defense mechanisms and strengthening cell wall factors (Collemare, O’Connell & Lebrun, 2019; Lo Presti et al., 2015; Pusztahelyi, Holb & Pocsi, 2015). By inducing penetration of the fungal cell, SMs are primarily engaged in biotrophic infection, causing infection to take place without killing the host. There has been an increase of SM production reported in genome and transcriptomic studies during different developmental stages of pathogen development in the host (Collemare, O’Connell & Lebrun, 2019; Keller, 2019; Rokas, Wisecaver & Lind, 2018).

Role of transporters

During infection, the transporter gene family was upregulated (Fig. 4). When rust pathogenesis is triggered, hyphae from haustoria which feed off the plant’s carbohydrates and amino acids at their active functional state (Ellis et al., 2009; El Gueddari et al., 2002; Voegele et al., 2001). Membrane transporters of M. larici-populina and P. graminis f. sp. tritici have homologs of the HXT1, AAT1, AAT2, and AAT3 transporters and H + ATPases from the bean rust pathogen U. fabae (Duplessis et al., 2011). Whenever a pathogen interacts with a host, all these transporters are upregulated. M. larici-populina and P. graminis f. sp. tritici exhibit higher levels of peptide uptake due to the presence of 22 and 21 oligopeptide membrane transporter (OPT) genes, respectively. Only 5-16 OPT genes were found in other basidiomycete fungi (Duplessis et al., 2011). OPT genes upregulated in planta (Duplessis et al., 2011) transport peptides released by inducible proteases (aspartic peptidase, subtilisin) once the leaf tissues are infected (Fig. 4). A reduction of the Major Facilitator Superfamily (MFS) is observed in M. larici-populina and P. graminis f. sp. tritici genomes when compared with other basidiomycetes (Duplessis et al., 2011). However, many MFS transcripts are upregulated in planta, such as HXT1 homologues. In the plant host expression of M. larici-populina and P. graminis f. sp. tritici, invertase genes are upregulated (Duplessis et al., 2011). Host hexoses, such as sucrose transporter Srt1, are typically used by invading rust pathogen hyphae (Voegele et al., 2001). There is no homologue for this sucrose transporter. During the invasion of rust fungi, membrane transporters play a critical role in providing the necessary fuel due to the high metabolic activity (Duplessis et al., 2011). It was also noticed that auxin efflux gene expression was much higher in rust fungi compared to other basidiomycetes (Duplessis et al., 2011). In U. maydis, auxin synthesis gene homologs are upregulated during infection of the host (Turian & Hamilton, 1960; Basse et al., 1996; Reineke et al., 2008). The growth of plants depends on auxin synthesis while pathogen auxins are crucial for host signaling, defense strategies, or plant cell wall integrity during rust infections.

Role of carbohydrate-active enzymes

In addition to proteases, lipases, and sugar-cleaving enzymes, a variety of carbohydrate-active enzymes (CAZymes) are up-regulated (Fig. 4) in rust pathogen plants (Cantarel et al., 2009; Duplessis et al., 2011). This suggests that the pathogen uses degradative enzymes and fungal hyphae to penetrate and enter the host cell. Upon comparing 21 sequenced fungi, it was found that glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), and carbohydrate esterases (CE) (Duplessis et al., 2011) are similar to those found in the basidiomycete symbiont, L. bicolor (Martin et al., 2008), but are less numerous than hemibiotrophs and necrotrophs (e.g., Magnaporthe oryzae), and saprotrophs (including Neurospora crassa; Coprinopsis cinerea; Schizophyllum commune) (Ohm et al., 2010). The biotroph U. maydis does not contain many CAZymes (100 members) (Kämper et al., 2006). The evolution of a biotrophic lifestyle in rust fungi resulted in the loss of secreted hydrolytic GH and PL enzymes known to interact with plant cell wall polysaccharides (Duplessis et al., 2011). Evolution to the biotrophic lifestyle also led to the loss of cellulose binding carbohydrate-binding module 1 (CBM1) (Fig. 4). The GHs that cleave plant celluloses and hemicelluloses are moderately upregulated (e.g., GH7, GH10, GH12, GH26, and GH27) in comparison with biotrophs U. maydis or M. oryzae (which is the hemibiotroph). Plants with these upregulated enzymes have also shown that presence of α-mannosidase (GH47) and β-1,3-glucanase (GH5) transcripts (Duplessis et al., 2011) are involved with colonization or penetration of parenchymal cells. The cell wall of a fungus is reconstructed and altered to disguise invading hypha from the host when there is an infection (El Gueddari et al., 2002) by chitin deacetylases (CE4), something which is also seen in P. graminis f. sp. tritici, M. laricipopulina, and the symbiont L. bicolor (Martin et al., 2008).

Nitrate and sulfate assimilation pathway deficiencies in rust fungi

Rust fungi, like other obligate pathogens, cannot grow in vitro. Hence, M. larici-populina and P. graminis f. sp. tritici are unlikely to carry genes normally found in saprotrophic basidiomycetes. Several anabolic pathways have been scrutinized for possible deficiencies. NH4+ assimilation enzymes are present. However, nitrate assimilation genes were not present in either rust pathogen genome. Nitrate/nitrite porters and nitrite reductases were not found in other fungis’ gene clusters associated with nitrate assimilation (Slot & Hibbett, 2007). Sulfate assimilation genes were found in M. larici-populina, but not in P. graminis f. sp. tritici. SiR subunits, α- and β of sulfite reductase, were absent in the latter fungus, whereas the M. larici-populina β-subunit of SiR has no transketolase domain similar to the SiRs found in other fungal systems. Both rust fungi have dysfunctional nitrate and sulfate assimilation pathways that can be related to obligate biotrophs. This is because they need minimal nitrogen sources (different amino acids and ammonium ion) and there is no uptake of sulfur from the plant system (Duplessis et al., 2011). The same metabolic deficiencies (Fig. 3) have been discovered in different plant pathogens from different evolutionary lineages, one belonging to the oomycete (H. arabidopsis) and the other to the ascomycete (B. graminis) (Spanu et al., 2010; Baxter et al., 2010).

Role of signal molecules in obligate parasitism

A new hypothesis suggests signal molecules coming from the host plant helps in an obligate biotrophic lifestyle through the regulation of metabolic gene expressions. (Fig. 3). Infection specific organs, like haustoria and appressoria (specialized structures for nutrient uptake and entry respectively), develop in response to plant signals (Hamer & Talbot, 1998; Tucker & Talbot, 2001). In plants, for example, rusts recognize the surface of the cell wall, prompting hyphae to form (Staples, 2000). In Magnaporthe, hydrophobic surfaces cause appressoria to develop (Talbot et al., 1996; Hamer & Talbot, 1998). Products known to degrade cutin cause appressoria formation in Blumeria (Both & Spanu, 2004). It is possible that this essential factor controls the essential metabolic machinery of biotrophs involved in nutrient uptake and utilization. The fungus must reveal its transporters and pumps at the exact moment, at the right locations, and at the correct intensity to survive. Plant derived stimuli, rather than feeding structures, are essential to survival. Furthermore, during their life cycle, it is these stimuli that catalyze the catabolic and anabolic reactions necessary for growth and nutrition of biotrophs. At the moment, the alternative hypothesis is supported by some direct evidence. A variety of metabolic genes are expressed during both development and pathogen attack in barley powdery mildew (Both et al., 2005), as well as functional characteristics associated with uptake in rust haustoria (Sohn et al., 2000; Voegele et al., 2001; Jakupovic et al., 2006). In arbuscular mycorrhizal fungi, intraradical mycelium was found to contain fatty acid synthase activity (Trepanier et al., 2005), and mycelium associated with intraradical and extraradical growth exhibit differential expression of genes related to nitrogenous compound metabolism (Govindarajulu et al., 2005). Based on this new hypothesis, the expression pattern displayed during pathogen germination should be disrupted when fungi are observed to partially grow in vitro. Another test will determine if there are deficiencies or mutations in the regulatory elements (e.g., promoters) that control metabolic genes. The whole genomes of obligate and non-obligate related fungi can be compared to unravel this information. There may be a connection between evolutionary radiation and the spontaneous development and modifications in regulatory mechanisms of genes which bring about a major change in the expression pattern (Gilad et al., 2006).