Magnaporthe oryzae (M. oryzae), the causative fungus of rice blast disease, has the ability to (and does) destroy large swathes of cereal crops world-wide. Quite a long time ago we wrote about the possibility of using CRISPR to help control the disease by manipulating rice-plant genes to decrease susceptibility to the disease. The effort to increase resistance to rice blast was also mentioned in our post about the current status of genetic engineering’s promise to assist sustainable control of plant disease and our post on the possibility of increasing resistance through increased expression of the gene WRKY45.
Although progress towards increasing resistance to the disease is being made, better understanding the pathogen’s methods of infection and spread in turn informs methods of controlling and minimising the pathogen’s virulence.
The researchers behind a recent paper in Science delved into the methods of colonisation and spread used by the fungus on the background of previous research demonstrating that, once the fungus manages to invade the outer cuticle of the leaf, it suppresses plant immune responses. Concurrently, it produces hyphae that manage to spread to adjacent cells via a segment of constricted hyphae traversing plasmodesmata, rapidly infecting the tissue as result.
The Research Findings
The battery of tests performed by the researchers to better understand the fungus began with the use of transmission electron micrographs of rice sheath which confirmed that hyphae, normally measuring 5.0 µm in diameter, narrow to 0.6 µm in diameter to penetrate through the plasmodesmata, maintaining the integrity of the plasma membrane of the infected cells.
Figure 1A from article, showing the constriction of the invasive hyphae (IH) as it penetrates the rice cell wall (RCW).
Normally, callose is deposited to close plasmodesmata channels as an immune response in plants. This was visualised by the researchers but in cells that had been infected by the fungus, no callose was deposited 27 hours after infection. At 30 hours post infection, when initially invaded cells began dying, callose began to form at plasmodesmata but by this time the hyphae had entered adjacent cells. Callose then formed around the base of hyphae that had entered into the adjacent cell.
Testing the ability of the fungus to overcome the restriction on the size of molecules passing through plasmodesmata, mCherry expression vectors expressing either single or double-length mCherry proteins were inserted into infected and control rice tissue. In control tissue, single-length proteins were able to pass between cells while double-length proteins were excluded. However, in infected tissue, the double-length proteins were also able to pass between cells, indicating that the fungus is able to manipulate the size exclusion limit for long enough to assist hyphae spread. However, secreted effector proteins did not traverse the plasmodesmata into subsequently infected cells; effector proteins were limited to the initially infected host cell.
Figure 1G from article. Difference in movement of double mCherry proteins in plants infected and free from rice blast.
Investigating the control of hyphae growth, the researchers built upon earlier work that had shown that mutant variants of M. oryzae that knocked out a mitogen-activated protein kinase (Pmk1) failed to infect rice plants even when the plant leaves are wounded. To test this finding further, the authors of the paper created a Pmk1 allele that was sensitive to the analog 1NA-PP1, allowing the expression of Pmk1 to be manipulated in vivo. The study found that that when Pmk1 was turned off, the ability of the formed hyphae to constrict at the plasmodesmata entrance was reduced, preventing invasion of adjacent cells. What the researchers found was that in the presence of the analog the hyphae still formed terminal swellings at points where those hyphae met with the host cell walls but they were able to infect no further.
Part of figure 2A and figure 2C from article. Figure 2A demonstrates reduced ability for hyphae to invade adjacent cells when pmk1 expression is limited. Figure 2C highlights the swelling of hyphae when they meet the cell wall when pmk1 is inactive.
To confirm the role of the MAP kinase, a Green Flourescence Protein tag was added to the kinase protein to allow live imaging. Imaging showed that the gene was expressed at the time the fungus penetrates leaf cuticle and when the hyphae meet with plant cell walls just prior to those hyphae spreading to the adjacent cells.
RNA sequencing analysis was performed to compare gene expression levels when the Pmk1 gene was turned on and off. Inhibiting the gene resulted in the regulation of effector genes that are known to be involved in the suppression of plant immunity, as well as down-regulating a number of morphogenic regulators. To test whether it was the lack of host immunity suppression that was stopping hyphae from invading adjacent cells and not the Pmk1 kinase itself, the researchers suppressed host immunity simultaneously with Pmk1 suppression. What they found was even in the extreme test of killing plant tissue with ethanol and then rehydrating prior to inoculating the tissue with the fungus, suppression of Pmk1 expression still resulted in the hyphae being unable to infect adjacent cells, remaining trapped in the first host cell infected.
The result of these findings was the hypothesis that Pmk1 was critical to the fungus being able to constrict hyphae to cross plasmodesmata and invade neighbouring cells, the constriction being dependent on septin-dependent appressorium repolarisation, consistent with the effect Pmk1 gene expression had on the morphogenic regulators mentioned above.
To investigate the septin organisation effects of Pmk1 inhibition, a GFP tag was added to the Sep5 gene and imaged. What the researchers found was that the Sep5-GFP still accumulated where the hyphae met the cell wall but instead of forming septin collars at the site where the hyphae would cross the cell wall, the protein accumulated in a disorganised mass.
The results of these studies suggest that the Pmk1 MAP kinase pathway controls the ability of the fungus to use plasmodesmata as a means of moving hyphae from cell to cell, that ability relying on the constriction of the hyphae to a size that allows it to penetrate through these inter-cellular channels. Combined with the expression of effector molecules to suppress plant immunity that would otherwise see these channels blocked before the hyphae are able to invade, the pathway plays a significant role in the fungus’ virulence.
The conclusions drawn about the importance of the pathway in the fungus’ ability to infect and spread through rice plants must lead further research into methods of blocking the pathway in crops. Perhaps the use of RNAi targeted to this gene or host-derived endonucleases guided to the gene with CRISPR-based guide sequences could assist with application of this knowledge to a problem which regularly reduces the amount of rice produced in many parts of the world.